Blue-excitable water-solvated polymeric dyes

ABSTRACT

Water solvated polymeric dyes and polymeric tandem dyes having a blue excitation spectrum are provided. The polymeric dyes are conjugated polymers that can include a thiophene-containing co-monomer. The polymeric tandem dyes further include a signaling chromophore covalently linked to the conjugated polymer in energy-receiving proximity therewith. Also provided are labelled specific binding members that include the subject polymeric dyes. Methods of evaluating a sample for the presence of a target analyte and methods of labelling a target molecule in which the subject polymeric dyes find use are also provided. Systems and kits for practicing the subject methods are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §119 (e), this application claims priority to thefiling date of U.S. Provisional Patent Application Ser. No. 62/360,882,filed Jul. 11, 2016, the disclosure of which application is incorporatedherein by reference

INTRODUCTION

Fluorescent dyes are compounds which, when irradiated with light of awavelength which they absorb, emit light of a (usually) differentwavelength. Fluorescent dyes find use in a variety of applications inbiochemistry, biology and medicine, e.g. in diagnostic kits, inmicroscopy or in drug screening. Fluorescent dyes are characterized by anumber of parameters allowing a user to select a suitable dye dependingon the desired purpose. Parameters of interest include the excitationwavelength maximum, the emission wavelength maximum, the Stokes shift,the extinction coefficient, the fluorescence quantum yield and thefluorescence lifetime. Dyes may be selected according to the applicationof interest in order to, e.g., allow penetration of exciting radiationinto biological samples, to minimize background fluorescence and/or toachieve a high signal-to-noise ratio.

Molecular recognition involves the specific binding of two molecules.Molecules which have binding specificity for a target biomolecule finduse in a variety of research and diagnostic applications, such as thelabelling and separation of analytes, flow cytometry, in situhybridization, enzyme-linked immunosorbent assays (ELISAs), western blotanalysis, magnetic cell separations and chromatography. Targetbiomolecules may be detected by labelling with a fluorescent dye.

SUMMARY

Water solvated polymeric dyes and polymeric tandem dyes having a blueexcitation spectrum are provided. The polymeric dyes are conjugatedpolymers that can include a thiophene-containing co-monomer. Thepolymeric tandem dyes further include a signaling chromophore covalentlylinked to the conjugated polymer in energy-receiving proximitytherewith. Also provided are labelled specific binding members thatinclude the subject polymeric dyes. Methods of evaluating a sample forthe presence of a target analyte and methods of labelling a targetmolecule in which the subject polymeric dyes find use are also provided.Systems and kits for practicing the subject methods are also provided.

BRIEF DESCRIPTION OF THE FIGURES

It is understood that the drawings, described below, are forillustration purposes only. The drawings are not intended to limit thescope of the present teachings in any way.

FIG. 1 shows structures of exemplary polymeric dyes 1-7 of the presentdisclosure.

FIG. 2 illustrates the normalized absorption spectra of exemplary dyes1-7.

DEFINITIONS

Before describing exemplary embodiments in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used in the description.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Still, certain terms aredefined below for the sake of clarity and ease of reference.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. For example, the term “a primer”refers to one or more primers, i.e., a single primer and multipleprimers. It is further noted that the claims can be drafted to excludeany optional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

As used herein, the term “sample” relates to a material or mixture ofmaterials, in some cases in liquid form, containing one or more analytesof interest. In some embodiments, the term as used in its broadestsense, refers to any plant, animal or bacterial material containingcells or producing cellular metabolites, such as, for example, tissue orfluid isolated from an individual (including without limitation plasma,serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) orfrom in vitro cell culture constituents, as well as samples from theenvironment. The term “sample” may also refer to a “biological sample”.As used herein, the term “a biological sample” refers to a wholeorganism or a subset of its tissues, cells or component parts (e.g. bodyfluids, including, but not limited to, blood, mucus, lymphatic fluid,synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amnioticcord blood, urine, vaginal fluid and semen). A “biological sample” canalso refer to a homogenate, lysate or extract prepared from a wholeorganism or a subset of its tissues, cells or component parts, or afraction or portion thereof, including but not limited to, plasma,serum, spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors and organs. In certain embodiments, the sample hasbeen removed from an animal or plant. Biological samples may includecells. The term “cells” is used in its conventional sense to refer tothe basic structural unit of living organisms, both eukaryotic andprokaryotic, having at least a nucleus and a cell membrane. In certainembodiments, cells include prokaryotic cells, such as from bacteria. Inother embodiments, cells include eukaryotic cells, such as cellsobtained from biological samples from animals, plants or fungi.

As used herein, the terms “support bound” and “linked to a support” areused interchangeably and refer to a moiety (e.g., a specific bindingmember) that is linked covalently or non-covalently to a support ofinterest. Covalent linking may involve the chemical reaction of twocompatible functional groups (e.g., two chemoselective functionalgroups, an electrophile and a nucleophile, etc.) to form a covalent bondbetween the two moieties of interest (e.g. a support and a specificbinding member). In some cases, non-covalent linking may involvespecific binding between two moieties of interest (e.g., two affinitymoieties such as a hapten and an antibody or a biotin moiety and astreptavidin, etc.). In certain cases, non-covalent linking may involveabsorption to a substrate.

As used herein, the term “polypeptide” refers to a polymeric form ofamino acids of any length, including peptides that range from 2-50 aminoacids in length and polypeptides that are greater than 50 amino acids inlength. The terms “polypeptide” and “protein” are used interchangeablyherein. The term “polypeptide” includes polymers of coded and non-codedamino acids, chemically or biochemically modified or derivatized aminoacids, and polypeptides having modified peptide backbones in which theconventional backbone has been replaced with non-naturally occurring orsynthetic backbones. A polypeptide may be of any convenient length,e.g., 2 or more amino acids, such as 4 or more amino acids, 10 or moreamino acids, 20 or more amino acids, 50 or more amino acids, 100 or moreamino acids, 300 or more amino acids, such as up to 500 or 1000 or moreamino acids. “Peptides” may be 2 or more amino acids, such as 4 or moreamino acids, 10 or more amino acids, 20 or more amino acids, such as upto 50 amino acids. In some embodiments, peptides are between 5 and 30amino acids in length.

As used herein the term “isolated,” refers to an moiety of interest thatis at least 60% free, at least 75% free, at least 90% free, at least 95%free, at least 98% free, and even at least 99% free from othercomponents with which the moiety is associated with prior topurification.

A “plurality” contains at least 2 members. In certain cases, a pluralitymay have 10 or more, such as 100 or more, 1000 or more, 10,000 or more,100,000 or more, 10⁶ or more, 10⁷ or more, 10⁸ or more or 10⁹ or moremembers.

Numeric ranges are inclusive of the numbers defining the range.

As used herein, the term “specific binding” refers to the ability of acapture agent (or a first member of a specific binding pair) topreferentially bind to a particular analyte (or a second member of aspecific binding pair) that is present, e.g., in a homogeneous mixtureof different analytes. In some instances, a specific binding interactionwill discriminate between desirable and undesirable analytes in a samplewith a specificity of 10-fold or more for a desirable analyte over anundesirable analytes, such as 100-fold or more, or 1000-fold or more. Insome cases, the affinity between a capture agent and analyte when theyare specifically bound in a capture agent/analyte complex is at least10⁻⁸M, at least 10⁻⁹M, such as up to 10⁻¹⁰M.

As used herein, the terms “affinity” and “avidity” have the same meaningand may be used interchangeably herein. “Affinity” refers to thestrength of binding, increased binding affinity being correlated with alower Kd.

The methods described herein include multiple steps. Each step may beperformed after a predetermined amount of time has elapsed betweensteps, as desired. As such, the time between performing each step may be1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds ormore, 5 minutes or more, 10 minutes or more, 60 minutes or more andincluding 5 hours or more. In certain embodiments, each subsequent stepis performed immediately after completion of the previous step. In otherembodiments, a step may be performed after an incubation or waiting timeafter completion of the previous step, e.g., a few minutes to anovernight waiting time.

As used herein, the terms “evaluating”, “determining,” “measuring,” and“assessing,” and “assaying” are used interchangeably and include bothquantitative and qualitative determinations.

The term “separating”, as used herein, refers to physical separation oftwo elements (e.g., by size or affinity, etc.) as well as degradation ofone element, leaving the other intact.

As used herein, the term “linker” or “linkage” refers to a linkingmoiety that connects two groups and has a backbone of 100 atoms or lessin length. A linker or linkage may be a covalent bond that connects twogroups or a chain of between 1 and 100 atoms in length, for example achain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbonatoms in length, where the linker may be linear, branched, cyclic or asingle atom. In some cases, the linker is a branching linker that refersto a linking moiety that connects three or more groups. In certaincases, one, two, three, four or five or more carbon atoms of a linkerbackbone may be optionally substituted with a sulfur, nitrogen or oxygenheteroatom. In some cases, the linker backbone includes a linkingfunctional group, such as an ether, thioether, amino, amide,sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioesteror imine. The bonds between backbone atoms may be saturated orunsaturated, and in some cases not more than one, two, or threeunsaturated bonds are present in a linker backbone. The linker mayinclude one or more substituent groups, for example with an alkyl, arylor alkenyl group. A linker may include, without limitations,polyethylene glycol; ethers, thioethers, tertiary amines, alkyls, whichmay be straight or branched, e.g., methyl, ethyl, n-propyl,1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl(t-butyl), and the like. The linker backbone may include a cyclic group,for example, an aryl, a heterocycle or a cycloalkyl group, where 2 ormore atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included inthe backbone. A linker may be cleavable or non-cleavable.

As used herein, the terms “polyethylene oxide”, “PEO”, “polyethyleneglycol” and “PEG” are used interchangeably and refer to a polymericgroup including a chain described by the formula —(CH₂-CH₂—O—)_(n)— or aderivative thereof. In some embodiments, “n” is 5000 or less, such as1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 40 orless, 30 or less, 20 or less, 15 or less, such as 3 to 15, or 10 to 15.It is understood that the PEG polymeric group may be of any convenientlength and may include a variety of terminal groups and/or furthersubstituent groups, including but not limited to, alkyl, aryl, hydroxyl,amino, acyl, acyloxy, and amido terminal and/or substituent groups. PEGgroups that may be adapted for use in the subject multichromophoresinclude those PEGs described by S. Zalipsky in “Functionalizedpoly(ethylene glycol) for preparation of biologically relevantconjugates”, Bioconjugate Chemistry 1995, 6 (2), 150-165; and by Zhu etal in “Water-Soluble Conjugated Polymers for Imaging, Diagnosis, andTherapy”, Chem. Rev., 2012, 112 (8), pp 4687-4735.

As used herein, the term “alkyl” by itself or as part of anothersubstituent refers to a saturated branched or straight-chain monovalenthydrocarbon radical derived by the removal of one hydrogen atom from asingle carbon atom of a parent alkane. Alkyl groups of interest include,but are not limited to, methyl; ethyl, propyls such as propan-1-yl orpropan-2-yl; and butyls such as butan-1-yl, butan-2-yl,2-methyl-propan-1-yl or 2-methyl-propan-2-yl. In some embodiments, analkyl group includes from 1 to 20 carbon atoms. In some embodiments, analkyl group includes from 1 to 10 carbon atoms. In certain embodiments,an alkyl group includes from 1 to 6 carbon atoms, such as from 1 to 4carbon atoms. This term includes, by way of example, linear and branchedhydrocarbyl groups such as methyl (CH₃—), ethyl (CH₃CH₂—), n-propyl(CH₃CH₂CH₂—), isopropyl ((CH₃)₂CH—), n-butyl (CH₃CH₂CH₂CH₂—), isobutyl((CH₃)₂CHCH₂—), sec-butyl ((CH₃)(CH₃CH₂)CH—), t-butyl ((CH₃)₃C—),n-pentyl (CH₃CH₂CH₂CH₂CH₂—), and neopentyl ((CH₃)₃CCH₂—).

The term “substituted alkyl” refers to an alkyl group as defined hereinwherein one or more carbon atoms in the alkyl chain have been optionallyreplaced with a heteroatom such as —O—, —N—, —S—, —S(O)_(n)— (where n is0 to 2), —NR— (where R is hydrogen or alkyl) and having from 1 to 5substituents selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-aryl,—SO₂-heteroaryl, and —NR^(a)R^(b), wherein R′ and R″ may be the same ordifferent and are chosen from hydrogen, optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl andheterocyclic.

“Alkoxy” refers to the group —O-alkyl, wherein alkyl is as definedherein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. Theterm “alkoxy” also refers to the groups alkenyl-O—, cycloalkyl-O—,cycloalkenyl-O—, and alkynyl-O—, where alkenyl, cycloalkyl,cycloalkenyl, and alkynyl are as defined herein.

The term “substituted alkoxy” refers to the groups substituted alkyl-O—,substituted alkenyl-O—, substituted cycloalkyl-O—, substitutedcycloalkenyl-O—, and substituted alkynyl-O— where substituted alkyl,substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyland substituted alkynyl are as defined herein.

“Amino” refers to the group —NH₂.

The term “substituted amino” refers to the group —NRR where each R isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,substituted alkynyl, aryl, heteroaryl, and heterocyclyl provided that atleast one R is not hydrogen.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon radical derived by the removal of onehydrogen atom from a single carbon atom of an aromatic ring system. Arylgroups of interest include, but are not limited to, groups derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, coronene, fluoranthene, fluorene, hexacene,hexaphene, hexalene, as-indacene, s-indacene, indane, indene,naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene,trinaphthalene and the like. In certain embodiments, an aryl groupincludes from 6 to 20 carbon atoms. In certain embodiments, an arylgroup includes from 6 to 12 carbon atoms. Examples of an aryl group arephenyl and naphthyl.

“Heteroaryl” by itself or as part of another substituent, refers to amonovalent heteroaromatic radical derived by the removal of one hydrogenatom from a single atom of a heteroaromatic ring system. Heteroarylgroups of interest include, but are not limited to, groups derived fromacridine, arsindole, carbazole, β-carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, triazole, benzotriazole, thiophene,triazole, xanthene, benzodioxole and the like. In certain embodiments,the heteroaryl group is from 5-20 membered heteroaryl. In certainembodiments, the heteroaryl group is from 5-10 membered heteroaryl. Incertain embodiments, heteroaryl groups are those derived from thiophene,pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline,imidazole, oxazole and pyrazine.

“Heterocycle,” “heterocyclic,” “heterocycloalkyl,” and “heterocyclyl”refer to a saturated or unsaturated group having a single ring ormultiple condensed rings, including fused bridged and spiro ringsystems, and having from 3 to 20 ring atoms, including 1 to 10 heteroatoms. These ring atoms are selected from the group consisting ofnitrogen, sulfur, or oxygen, wherein, in fused ring systems, one or moreof the rings can be cycloalkyl, aryl, or heteroaryl, provided that thepoint of attachment is through the non-aromatic ring. In certainembodiments, the nitrogen and/or sulfur atom(s) of the heterocyclicgroup are optionally oxidized to provide for the N-oxide, —S(O)—, or—SO₂-moieties.

Examples of heterocycles and heteroaryls include, but are not limitedto, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine,tetrahydrofuranyl, and the like.

Unless otherwise constrained by the definition for the heterocyclicsubstituent, such heterocyclic groups can be optionally substituted with1 to 5, or from 1 to 3 substituents, selected from alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl,—SO₂-heteroaryl, and fused heterocycle.

The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl andsubstituted alkylene-aryl where alkylene, substituted alkylene and arylare defined herein.

“Alkylene” refers to divalent aliphatic hydrocarbyl groups preferablyhaving from 1 to 6 and more preferably 1 to 3 carbon atoms that areeither straight-chained or branched, and which are optionallyinterrupted with one or more groups selected from —O—, —NR¹⁰—,—NR¹⁰C(O)—, —C(O)NR¹⁰— and the like. This term includes, by way ofexample, methylene (—CH₂—), ethylene (—CH₂CH₂—), n-propylene(—CH₂CH₂CH₂—), iso-propylene (—CH₂CH(CH₃)—), (—C(CH₃)₂CH₂CH₂—),(—C(CH₃)₂CH₂C(O)—), (—C(CH₃)₂CH₂C(O)NH—), (—CH(CH₃)CH₂—), and the like.“Substituted alkylene” refers to an alkylene group having from 1 to 3hydrogens replaced with substituents as described for carbons in thedefinition of “substituted” below.

“Substituted” refers to a group in which one or more hydrogen atoms areindependently replaced with the same or different substituent(s).Substituents of interest include, but are not limited to, alkylenedioxy(such as methylenedioxy), —M, —R⁶⁰, —O⁻, ═O, —OR⁶⁰, —SR⁶⁰, —S⁻, ═S,—NR⁶⁰R⁶¹, ═NR⁶⁰, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O⁻,—S(O)₂OH, —S(O)₂R⁶⁰, —OS(O)₂O^(—, —OS(O)) ₂R⁶⁰, —P(O)(O⁻)₂, —P(O)(OR⁶⁰)(O⁻), —OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰, —C(S)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹,—C(O)O⁻, —C(S) OR⁶⁰, —NR⁶²C(O)NR⁶⁰R⁶¹, —NR⁶²C(S)NR⁶⁰R⁶¹,—NR⁶²C(NR⁶³)NR⁶⁰R⁶¹ and —C(NR⁶²)NR⁶⁰R⁶¹ where M is halogen; R⁶⁰, R⁶¹,R⁶² and R⁶³ are independently hydrogen, alkyl, substituted alkyl,alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl,heteroaryl or substituted heteroaryl, or optionally R⁶⁰ and R⁶¹ togetherwith the nitrogen atom to which they are bonded form a cycloheteroalkylor substituted cycloheteroalkyl ring; and R⁶⁴ and R⁶⁵ are independentlyhydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substitutedcycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl,substituted aryl, heteroaryl or substituted heteroaryl, or optionallyR⁶⁴ and R⁶⁵ together with the nitrogen atom to which they are bondedform a cycloheteroalkyl or substituted cycloheteroalkyl ring. In certainembodiments, substituents include —M, —R⁶⁰ ═O, —OR⁶⁰, —SR⁶⁰, ═S,—NR⁶⁰R⁶¹, ═NR⁶⁰, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)₂R⁶⁰,—OS(O)₂O⁻, —OS(O)₂R⁶⁰, —P(O)(O⁻)₂, —P(O)(OR⁶⁰)(O⁻), —OP(O)(OR⁶⁰)( OR⁶¹),—C(O)R⁶⁰, —C(S)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, —C(O)O⁻, —NR⁶²C(O)NR⁶⁰R⁶¹.In certain embodiments, substituents include —M, —R⁶⁰, ═O, —OR⁶⁰,—NR⁶⁰R⁶¹, —CF₃, —CN, —NO₂, —S(O)₂R⁶⁰, —P(O)(OR⁶⁰)(O⁻), —OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰, —C(O)OR⁶⁰, —C(O)NR⁶⁰R⁶¹, —C(O)O⁻. In certainembodiments, substituents include —M, —R⁶⁰ , ═O, —OR⁶⁰, —SR⁶⁰, —NR⁶⁰R⁶¹,—CF₃, —CN, —NO₂, —S(O)₂R⁶⁰, —OP(O)(OR⁶⁰)(OR⁶¹), —C(O)R⁶⁰, —C(O)OR⁶⁰ ,—C(O)O⁻, where R⁶⁰, R⁶¹ and R⁶² are as defined above. For example, asubstituted group may bear a methylenedioxy substituent or one, two, orthree substituents selected from a halogen atom, a (1-4C)alkyl group anda (1-4C)alkoxy group. When the group being substituted is an aryl orheteroaryl group, the substituent(s) (e.g., as described herein) may bereferred to as “aryl substituent(s)”.

Other definitions of terms may appear throughout the specification.

DETAILED DESCRIPTION

As summarized above, water solvated polymeric dyes and polymeric tandemdyes having a blue excitation spectrum are provided. The polymeric dyescan be conjugated polymers that include a thiophene-containingco-monomer. The polymeric tandem dyes further include a signalingchromophore covalently linked to the multichromophore inenergy-receiving proximity therewith. Also provided are labelledspecific binding members that include the subject polymeric dyes.Methods of evaluating a sample for the presence of a target analyte andmethods of labelling a target molecule in which the subject polymericdyes find use are also provided. Systems and kits for practicing thesubject methods are also provided.

Before the various embodiments are described in greater detail, it is tobe understood that the teachings of this disclosure are not limited tothe particular embodiments described, and as such can, of course, vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present teachings will be limitedonly by the appended claims.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way. While the present teachings are described in conjunction withvarious embodiments, it is not intended that the present teachings belimited to such embodiments. On the contrary, the present teachingsencompass various alternatives, modifications, and equivalents, as willbe appreciated by those of skill in the art.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present teachings, some exemplarymethods and materials are now described.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentclaims are not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided can be differentfrom the actual publication dates which can be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentteachings. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

All patents and publications, including all sequences disclosed withinsuch patents and publications, referred to herein are expresslyincorporated by reference.

In further describing the subject invention, polymeric dyes includinglight harvesting multichromophores and related polymeric tandem dyesfurther including a signaling chromophore are described first in greaterdetail. Next, labelled specific binding members which include thesubject polymeric dyes are described. Then, methods of interest in whichthe subject polymeric dyes find use are reviewed. Systems and kits thatmay be used in practicing methods of the present disclosure are alsodescribed.

Polymeric Dyes

As summarized above, the present disclosure provides water solvatedpolymeric dyes having a blue excitation spectrum. The subject polymericdyes can be pi-conjugated polymers including thiophene-basedco-monomers. The polymer can be an alternating co-polymer or a randomco-polymer of co-monomers. In some instances, the subject polymeric dyesprovide narrow absorption peak width relative to other dyes of interest.In multi-color applications, being able to use multiple excitationsources is critical to achieving multiplicity and a high number ofparameters. As peak widths narrow, the amount of excitation by sourcesother than the wavelength intended decreases. In addition to narrow peakwidth, minimizing absorption at other common excitation sources iscritical for higher parameter experiments. The degree of cross-sourceexcitation can be tuned for the subject polymeric dyes by selection of aparticular structure, co-monomers, and mixtures of co-blocks.

The subject polymeric dyes feature termini on the conjugated polymerchains that can include a free chemoselective functional group thatprovides for bioconjugation. In some cases, such functionality isreferred to as an end linker. With these end linkers, a covalent bondcan be formed to attach a biomolecule such as an antibody, apolynucleotide, or aptamer. For example, polymeric dye-labeledantibodies find use in flow cytometry as reagents exhibiting highbrightness. Additionally, orthogonal chemoselective functional groupscan be installed along the conjugated polymer chain that can be used foreither bioconjugation or the attachment of acceptor signalingchromophores in donor-acceptor polymeric tandem dyes.

As used herein, the terms “light harvesting multichromophore”,“polymeric dye” and “conjugated polymer” are used interchangeably andrefer to a conjugated polymer which has a structure capable ofharvesting light with a particular absorption maximum wavelength andconverting it to emitted light at a longer emission maximum wavelength.In some cases, the light harvesting multichromophore is itselffluorescent. Conjugated polymers (CPs) are characterized by adelocalized electronic structure and may have an effective conjugationlength that is substantially shorter than the length of the polymerchain, because the backbone may contain a large number of conjugatedsegments in close proximity. In some cases, conjugated polymers areefficient for light harvesting and provide for optical amplification viaForster energy transfer to an acceptor.

As used herein the term “unit” refers to a structural subunit of apolymer. The term unit is meant to include monomers, co-monomers,co-blocks, conjugated segments, repeating units, and the like. A“repeating unit” is a subunit of a polymer that is defined by theminimum number of distinct structural features that are required for theunit to be considered monomeric, such that when the unit is repeated ntimes, the resulting structure describes the polymer or a block thereof.In some cases, the polymer may include two or more different repeatingunits, e.g., when the polymer is a multiblock polymer or a randomarrangement of units, each block may define a distinct repeating unit,e.g., an n-block and a m-block. It is understood that a variety ofarrangements of n and/or m repeating units or blocks are possible andthat in the depicted formula of the subject multichromophores describedherein any convenient linear arrangements of n and m co-blocks ofvarious lengths are included within the structure of the overallpolymer. In some cases, a repeating unit of the polymer includes asingle monomer group. In certain instances, a repeating unit of thepolymer includes two or more monomer groups, i.e., co-monomer groups,such as two, three, four or more co-monomer groups. As used herein, theterm “co-monomer” or “co-monomer group” refers to a structural unit of apolymer that may itself be part of a repeating unit of the polymer. Insome embodiments, the conjugated polymer includes a block copolymer thatis composed of blocks of polymerized monomers. In such cases, the blockcopolymer may be described as having distinct repeating units eachcorresponding to a distinct co-block of the polymer. In some cases, thepolymer is a diblock copolymer that contains two different co-blocks. Insuch cases, the polymer may be described as including co-blocks, whereeach co-block may be composed of co-monomers, such as one, two, three ormore co-monomers.

The multichromophore may have any convenient length. In some cases, theparticular number of monomeric repeating units or segments of themultichromophore may fall within the range of 2 to 500,000, such as 2 to100,000, 2 to 30,000, 2 to 10,000, 2 to 3,000 or 2 to 1,000 units orsegments, or such as 5 to 100,000, 10 to 100,000, 100 to 100,000, 200 to100,000, or 500 to 50,000 units or segments. In some instances, theparticular number of monomeric repeating units or segments of themultichromophore may fall within the range of 2 to 1,000, such as 2 to500, 2 to 100, 3 to 100, 4 to 100, 5 to 100, 6 to 100, 7 to 100, 8 to100, 9 to 100 or 10 to 100 units or segments.

The multichromophore may be of any convenient molecular weight (MW). Insome cases, the MW of the multichromophore may be expressed as anaverage molecular weight. In some instances, the polymeric dye has anaverage molecular weight in the range of 500 to 500,000, such as from1,000 to 100,000, from 2,000 to 100,000, from 10,000 to 100,000 or evenan average molecular weight in the range of 50,000 to 100,000.

The subject polymeric dyes can include a certain minimum content ofthiophene-containing co-monomers that can provide for a desirablespectroscopic property of interest. The content (e.g., mol % values) ofthiophene-containing co-monomers in the polymeric dyes (e.g., asdescribed herein) can be measured and referred to according to aparticular thiophene-containing co-monomer or according to thecumulative total of all thiophene-containing co-monomers in thepolymeric dyes. In some embodiments, the multichromophore includes athiophene-containing co-monomer(s) that constitutes 5% or more bymolarity (e.g., 5 mol %) of the multichromophore, such as 10% or more,15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% ormore, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more,70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or evenmore by molarity of the multichromophore. In such cases, themultichromophore may include 5 or more repeating units, such as 10 ormore, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 ormore, 80 or more, 90 or more, 100 or more, 200 or more, 500 or more,1000 or more, 10,000 or more, or even more repeating units. In suchcases, the multichromophore may include 5 or more co-monomer units, suchas 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 ormore, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 500or more, 1000 or more, 10,000 or more, or even more co-monomer units. Incertain embodiments, the thiophene-containing co-monomer(s) of interestconstitutes 25% or more by molarity of the multichromophore, such as 30%or more, 40% or more, 45% or more, 50% or more, or even more by molarityof the multichromophore, which includes 5 or more repeating units, suchas 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 ormore, 70 or more, 80 or more, 90 or more, 100 or more repeating units.

In some embodiments, the multichromophore includes a plurality of firstoptically active units forming a conjugated system, having an absorptionwavelength (e.g., as described herein) at which the first opticallyactive units absorb light to form an excited state. In certaininstances, the multichromophore includes a conjugated polymer segment oran oligomeric structure including bandgap-lowering n-conjugatedrepeating units.

The subject multichromophore may have one or more desirablespectroscopic properties, such as a particular absorption maximumwavelength, a particular emission maximum wavelength, extinctioncoefficient, quantum yield, and the like. The subject polymeric dyes andpolymeric tandem dyes provide for a variety of absorption and emissionprofiles which depend on a variety of factors such as the selectedco-monomers, linking groups, substituents and optional linked signalingchromophores of which the polymers are composed.

The subject water solvated polymeric dyes have a blue excitationspectrum. As used herein, a blue excitation spectrum is an excitationspectrum having a full width at half maximum (FWHM) located in the rangeof 375 nm to 575 nm. Full width at half maximum (FWHM) is an expressionof the extent of a function given by the difference between the twoextreme values of the independent variable at which the dependentvariable is equal to half of its maximum value. In other words, it isthe width (a difference or range of two particular wavelengths) of anexcitation spectrum curve measured between those points on the y-axiswhich are half the maximum amplitude. In some instances, the FWHM can bereferred to as the difference between the wavelengths. In someinstances, the FWHM can be referred to in reference to a particularregion of the spectrum within which the wavelengths defining the FWHMare located and measured.

In certain embodiments, the polymeric dye has a blue excitation spectrumhaving a full width at half maximum (FWHM) located in the range of 375nm to 550 nm. In certain embodiments, the polymeric dye has a blueexcitation spectrum having a full width at half maximum (FWHM) locatedin the range of 375 nm to 525 nm. In certain embodiments, the polymericdye has a blue excitation spectrum having a full width at half maximum(FWHM) located in the range of 375 nm to 500 nm. In certain embodiments,the polymeric dye has a blue excitation spectrum having a full width athalf maximum (FWHM) located in the range of 375 nm to 475 nm. In certainembodiments, the polymeric dye has a blue excitation spectrum having afull width at half maximum (FWHM) located in the range of 375 nm to 450nm. In certain embodiments, the polymeric dye has a blue excitationspectrum having a full width at half maximum (FWHM) located in the rangeof 400 nm to 575 nm. In certain embodiments, the polymeric dye has ablue excitation spectrum having a full width at half maximum (FWHM)located in the range of 410 nm to 575 nm. In certain embodiments, thepolymeric dye has a blue excitation spectrum having a full width at halfmaximum (FWHM) located in the range of 420 nm to 575 nm. In certainembodiments, the polymeric dye has a blue excitation spectrum having afull width at half maximum (FWHM) located in the range of 430 nm to 575nm. In certain embodiments, the polymeric dye has a blue excitationspectrum having a full width at half maximum (FWHM) located in the rangeof 440 nm to 575 nm. In certain embodiments, the polymeric dye has ablue excitation spectrum having a full width at half maximum (FWHM)located in the range of 450 nm to 575 nm. In certain embodiments, thepolymeric dye has a blue excitation spectrum having a full width at halfmaximum (FWHM) located in the range of 460 nm to 575 nm. In someinstances, the water solvated polymeric dyes have a blue excitationspectrum having a FWHM of 200 nm or less, such as 180 nm or less, 175 nmor less, 170 nm or less, 160 nm or less, 150 nm or less, 140 nm or less,130 nm or less, 120 nm or less, 110 nm or less, 100 nm or less, or evenless.

In some cases, the polymeric dye absorption maximum wavelength is in therange of 375 to 575 nm, such as 400 nm to 550 nm, 410 nm to 550 nm, 420nm to 540 nm, 430 nm to 540 nm or 440 nm to 530 nm. In certainembodiments, the multichromophore has an absorption maximum wavelengthof 550 nm or less, such as 525 nm or less, 500 nm or less, 490 nm orless, 480 nm or less, 470 nm or less, 460 nm or less, 450 nm or less,425 nm or less, or even less. In certain instances, the multichromophorehas an absorption maximum wavelength in the range of 375 nm to 440 nm.In certain instances, the multichromophore has an absorption maximumwavelength in the range of 400 nm to 450 nm. In certain instances, themultichromophore has an absorption maximum wavelength in the range of450 nm to 500 nm. In certain instances, the multichromophore has anabsorption maximum wavelength in the range of 500 nm to 550 nm. In someinstances, the polymeric dyes disclosed herein can provide an absorptionmaximum wavelength in the range from 440 nm to 530 nm. In certain cases,the polymeric dyes provide high extinction coefficients and high quantumyields that yield bright fluorescent reagents. In certain instances, thepolymeric dye has no yellow-green absorption. In some instances, thepolymeric dye has no absorption at 562 nm or greater, such as 570 nm orgreater, 580 nm or greater, 590 nm or greater, 600 nm or greater, 610 nmor greater, or 620 nm or greater.

In certain instances, the multichromophore has an absorption maximumwavelength of 380 nm±5 nm. In certain instances, the multichromophorehas an absorption maximum wavelength of 390 nm±5 nm. In certaininstances, the multichromophore has an absorption maximum wavelength of400 nm±5 nm. In certain instances, the multichromophore has anabsorption maximum wavelength of 410 nm±5 nm. In certain instances, themultichromophore has an absorption maximum wavelength of 420 nm±5 nm. Incertain instances, the multichromophore has an absorption maximumwavelength of 430 nm±5 nm. In certain instances, the multichromophorehas an absorption maximum wavelength of 440 nm±5 nm. In certaininstances, the multichromophore has an absorption maximum wavelength of450 nm±5 nm. In certain instances, the multichromophore has anabsorption maximum wavelength of 460 nm±5 nm. In certain instances, themultichromophore has an absorption maximum wavelength of 470 nm±5 nm. Incertain instances, the multichromophore has an absorption maximumwavelength of 480 nm±20 nm. In certain instances, the multichromophorehas an absorption maximum wavelength of 480 nm±10 nm. In certaininstances, the multichromophore has an absorption maximum wavelength of480 nm±5 nm. In certain instances, the multichromophore has anabsorption maximum wavelength of 490 nm±5 nm. In certain instances, themultichromophore has an absorption maximum wavelength of 500 nm±5 nm. Incertain instances, the multichromophore has an absorption maximumwavelength of 510 nm±5 nm. In certain instances, the multichromophorehas an absorption maximum wavelength of 520 nm±5 nm. In certaininstances, the multichromophore has an absorption maximum wavelength of530 nm±5 nm. In certain instances, the multichromophore has anabsorption maximum wavelength of 540 nm±5 nm. In certain instances, themultichromophore has an absorption maximum wavelength of 550 nm±5 nm. Incertain instances, the multichromophore has an absorption maximumwavelength of 560 nm±5 nm. In certain instances, the multichromophorehas an absorption maximum wavelength of 570 nm±5 nm.

In some embodiments, the multichromophore has an emission maximumwavelength in the range of 300 to 900 nm, such as 350 to 850 nm, 350 to600 nm, 360 to 500 nm, 370 to 500 nm, 380 to 500 nm, 390 to 500 nm or400 to 500 nm, where specific examples of emission maxima of interestinclude, but are not limited to: 395 nm ±5 nm, 460 nm±5 nm, 490 nm±5 nm,550 nm±5 nm, 560 nm±5 nm, 605 nm±5 nm, 650 nm±5 nm, 680 nm±5 nm, 700nm±5 nm, 805 nm±5 nm. In certain instances, the multichromophore has anemission maximum wavelength selected from the group consisting of 395nm, 460 nm, 490 nm, 550 nm, 560 nm, 605 nm, 650 nm, 680 nm, 700 nm and805 nm. In certain instances, the multichromophore has an emissionmaximum wavelength of 395 nm±5 nm. In some instances, themultichromophore itself has an emission maximum wavelength in the rangeof 375 to 900 nm (such as in the range of 380 nm to 900 nm, 390 nm to900 nm, or 400 nm to 900 nm).

In some instances, the multichromophore has an extinction coefficient of5×10⁵ cm⁻¹M⁻¹ or more, such as 6×10⁵ cm⁻¹M⁻¹ or more, 7×10⁵ cm⁻¹M⁻¹ ormore, 8×10⁵ cm⁻¹M⁻¹ or more, 9×10⁵ cm⁻¹M⁻¹ or more, such as 1×10⁶cm⁻¹M⁻¹ or more, 1.5×10⁶ cm⁻¹M⁻¹ or more, 2×10⁶ cm ⁻¹M⁻¹ or more, 2.5×10⁶ cm ⁻¹M⁻¹ or more, 3×10⁶ cm ⁻¹M⁻¹ or more, 4×10⁶ cm ⁻¹M⁻¹ or more,5×10⁶ cm ⁻¹M⁻¹ or more, 6×10⁶ cm ⁻¹M⁻¹ or more, 7×10⁶ cm⁻¹M⁻¹ or more,or 8×10⁶ cm ⁻¹M⁻¹ or more. In such cases, the multichromophore may have5 or more repeating units, such as 6 or more, 7 or more, 8 or more, 9 ormore, 10 or more, or even more repeating units. In some embodiments, themultichromophore has a molar extinction coefficient of 5×10⁵ M⁻¹ cm ⁻¹or more. In certain embodiments, the multichromophore has a molarextinction coefficient of 1×10⁶ M⁻¹ cm ⁻¹ or more.

In some instances, the multichromophore has an extinction coefficient of40,000 cm⁻¹M⁻¹ per repeating unit or more, such as 45,000 cm⁻¹M⁻¹ perrepeating unit or more, 50,000 cm⁻¹M⁻¹ per repeating unit or more,55,000 cm⁻¹M⁻¹ per repeating unit or more, 60,000 cm⁻¹M⁻¹ per repeatingunit or more, 70,000 cm⁻¹M⁻¹ per repeating unit or more, 80,000 cm⁻¹M⁻¹per repeating unit or more, 90,000 cm⁻¹M⁻¹ per repeating unit or more,100,000 cm⁻¹M⁻¹ per repeating unit or more, or even more. In someinstances, the 40,000 cm⁻¹M⁻¹ per repeating unit or more describedherein is an average extinction coefficient. In certain instances, therepeat unit of the multichromophore may include a single monomer, twoco-monomers, or three or more co-monomers. In some instances, themultichromophore has an extinction coefficient of 40,000 cm⁻¹M⁻¹ perco-monomer or more, such as 45,000 cm⁻¹M⁻¹ per co-monomer or more,50,000 cm⁻¹M⁻¹ per co-monomer or more, 55,000 cm⁻¹M⁻¹ per co-monomer ormore, 60,000 cm⁻¹M⁻¹ per co-monomer or more, 70,000 cm⁻¹M⁻¹ perco-monomer or more, 80,000 cm⁻¹M⁻¹ per co-monomer or more, 90,000cm⁻¹M⁻¹ per co-monomer or more, 100,000 cm⁻¹M⁻¹ per co-monomer or more,or even more. In some instances, the 40,000 cm⁻¹M⁻¹ per co-monomer ormore is an average extinction coefficient.

It is understood that in some cases the subject multichromophores mayinclude co-blocks (e.g., n and m co-blocks) or a random arrangement of nand m repeating units. The subject multichromophores may include anyconvenient linear arrangements of n and m co-blocks of various lengthswithin the structure of the overall polymer. In addition, themultichromophores may include any convenient arrangements of co-monomerswithin such n and/or m co-blocks. Unless indicated to the contrary, allpossible arrangements of co-monomers are meant to be included in thepolymeric dyes described herein. A variety of polymer synthesis methodsmay be utilized to prepare co-monomers and co-blocks of interest in thepreparation of the subject multichromophores. It is understood that insome cases, the polymerization methods may produce a compositionincluding a population of conjugated polymers that includes somevariation with respect to the particular length and/or terminal groups(i.e., end groups) present in each conjugated polymer of the population.The formulae depicted herein may refer to a single compound or to apopulation or sub-population of polymeric compounds. As used herein, *denotes a site for covalent attachment to the unsaturated backbone of aconjugated polymer.

In some embodiments, the subject polymeric dyes are pi-conjugatedpolymers including a thiophene-based co-monomer and an aryl orheteroaryl co-monomer. Aryl or heteroaryl co-monomers of interestinclude but are not limited to, fused tricyclic co-monomers, such asfluorene co-monomers, carbazole co-monomers, silole co-monomers orbridged biphenyl co-monomers. A fused tricyclic co-monomer is aco-monomer including a tricyclic aromatic group having three fused ringsin a configuration where two aryl or heteroaryl 6-membered rings arefused to a central 5 or 6-membered carbocyclic or heterocyclic ring. Insome cases, the fused tricyclic co-monomer includes two benzo or pyridorings fused to a central 5 or 6 membered carbocyclic or heterocyclicring. The fused tricyclic co-monomer can be pi-conjugated to adjacentco-monomers of a polymer backbone via any convenient ring atoms of thefused rings. The central 5- or 6-membered ring may be a carbocycle or aheterocycle, aromatic or partially saturated, and may further include asidechain substituent, e.g., a WSG and/or a linker to a chemoselectivetag. A bridged biphenyl co-monomer is a fused tricyclic co-monomerhaving a biphenyl group where the two phenyl rings are further linkedwith each other via a central 6 membered ring. In certain instances, thefused tricyclic co-monomer is described by the following structure:

where:

Y is C(R³)₂, —C(R³)₂C(R³)₂—, NR³, Si(R³)₂ or Se;

Z is CH, CR or N;

each R³ is independently selected from H, alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl,substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido,an aralkyl, a substituted aralkyl, a PEG moiety, -L¹-Z¹, where L¹ is alinker and Z¹ is a chemoslective tag (e.g., a tag including achemoslective functional group) and a WSG; and

each R is independently H or one or more aryl or heteroarylsubstituents. In some cases, each R refers to one or two ringsubstituents independently selected from halogen, sulfonate, alkoxy,substituted alkoxy, alkyl and substituted alkyl. In certain instances,at least two of Z in each ring is CH or CR. In certain instances, oneand only one of Z in each ring is N.

In certain instances, the fused tricyclic co-monomer is described by oneof the following structures:

where:

Y is C(R³)₂, —C(R³)₂C(R³)₂—, NR³, Si(R³)₂ or Se;

each R³ is independently selected from H, alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl,substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido,an aralkyl, a substituted aralkyl, a PEG moiety, -L¹-Z¹, where L¹ is alinker and Z¹ is a chemoslective tag (e.g., a tag including achemoslective functional group) and a WSG; and

each R is independently H, R³ or one or more aryl or heteroarylsubstituents. In some cases, each R refers to one or two ringsubstituents independently selected from halogen, sulfonate, alkoxy,substituted alkoxy, alkyl and substituted alkyl. The symbol “* ” denotesa site for covalent attachment to the unsaturated backbone of aconjugated polymer, e.g., a π conjugated segment, a terminal group, alinker and a linked specific binding member. It is understood that forany of the formulae described herein which includes a 2,7-pi-conjugatedfused tricyclic co-monomer, an analogous formula including an analogous3,6-pi-conjugated co-monomer could also be depicted. In certain cases,the fused tricyclic co-monomer is a fluorene co-monomer where Y isC(R³)₂. In some cases, the fused tricyclic co-monomer is a carbazoleco-monomer where Y is NR³. In some cases, the fused tricyclic co-monomeris a silole co-monomer where Y is Si(R³)₂. In some cases, the fusedtricyclic co-monomer is a bridged biphenyl co-monomer where Y is—C(R³)₂C(R³)₂—. In some cases, the fused tricyclic co-monomer is abridged biphenyl co-monomer where Y is —CHR³CHR³—. In certain instancesof any of the fused tricyclic co-monomers described herein, each R isindependently selected from H, halogen, alkoxy, substituted alkoxy,alkyl and substituted alkyl. In certain cases, each R is independentlyselected from H, fluoro, chloro, methoxy, substituted alkoxy, alkyl andsubstituted alkyl.

In certain instances, the fused tricyclic co-monomer is described by oneof the following structures:

wherein:

each R³ is independently selected from H, alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl,substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido,an aralkyl, a substituted aralkyl, a PEG moiety, -L¹-Z¹, where L¹ is alinker and Z¹ is a chemoslective tag (e.g., a tag including achemoslective functional group) and a WSG; and

each R is independently H, R³ or one or more aryl or heteroarylsubstituents. In some cases, each R refers to one or two ringsubstituents independently selected from halogen, sulfonate, alkoxy,substituted alkoxy, alkyl and substituted alkyl. In some cases, each Ris fluoro or methoxy. In some cases, at least one R³ is a WSG. In somecases, at least one R³ is -L¹-Z¹.

In some instances, the multichromophore includes a conjugated segmenthaving the structure of formula (I):

wherein:

M¹, M² and M³ are independently an aryl or heterocyclic co-monomerwherein at least one of M¹, M² and M³ is a thiophene containingco-monomer;

Z¹ is a chemoselective functional group or a linked signalingchromophore;

Z⁵ and Z⁶ are independently CR or N where R is selected from H, halogen,alkoxy, substituted alkoxy, alkyl and substituted alkyl;

G¹ and G² are each independently selected from a terminal group, a πconjugated segment, a linker and a linked specific binding member;

Y¹, Y² and Y³ are independently C(R³)₂, —C(R³)₂C(R³)₂—, NR³, Si(R³)₂ orSe;

each R³ is independently selected from H, alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl,substituted acyl, alkoxy, substituted alkoxy, amido, substituted amidoand a WSG;

a-i are independently 0 or 1;

each n, m and o are independently 0 or an integer from 1 to 10,000; and

p is an integer from 1 to 100,000. In some instances of formula (I), oneof co-monomers M² and M³ includes a linked chemoselective functionalgroup or a linked signaling chromophore.

In certain embodiments of formula (I), Y¹ is C(R³)₂. In some embodimentsof formula (I), Y¹ is NR³. In certain instances of formula (I), Y¹ isSi(R³)₂. In certain cases of formula (I), Y¹ is —C(R³)₂C(R³)₂—. In somecases of formula (I), Y¹ is —CHR³CHR³—. In certain cases of formula (I),Y¹ is Se. In certain embodiments of formula (I), Y² is C(R³)₂. In someinstances of formula (I), Y² is NR³. In certain embodiments of formula(I), Y² is Si(R³)₂. In certain cases of formula (I), Y² is—C(R³)₂C(R³)₂—. In some cases of formula (I), Y² is —CHR³CHR³—. In someembodiments of formula (I), Y² is Se. In certain instances of formula(I), Y³ is C(R³)₂ wherein one R³ comprises Z¹. In some cases of formula(I), Y³ is NR³ wherein R³ comprises Z¹. In certain cases of formula (I),Y³ is Si(R³)₂ wherein one R³ comprises Z¹. In certain cases of formula(I), Y³ is —C(R³)₂C(R³)₂—, wherein one R³ comprises Z¹. In some cases offormula (I), Y³ is —CHR³CHR³— wherein one R³ comprises Z¹. In certainembodiments of formula (I), Z¹ is a chemoselective functional group. Incertain cases of formula (I), Z¹ is a linked signaling chromophore. Incertain embodiments of formula (I), at least one of G¹ and G² is alinker. In certain embodiments of formula (I), at least one of G¹ and G²is a linked specific binding member.

In some instances of formula (I), each fused tricyclic co-monomer is ofthe same core structure, e.g., a fluorene, a carbazole, a silole or abridged bi-phenyl co-monomer. In some instances of formula (I), eachfused tricyclic co-monomer is independently selected from a fluorene, acarbazole, a silole and a bridged bi-phenyl co-monomer. In certaininstances of a fused tricyclic co-monomer of formula (I), one of Z⁵ andZ⁶ is CR. In some instances of a fused tricyclic co-monomer of formula(I), both of Z⁵ and Z⁶ are independently CR. In some embodiments of afused tricyclic co-monomer of formula (I), Z⁵ is CR and Z⁶ is N. Incertain cases of a fused tricyclic co-monomer of formula (I), Z⁵ is Nand Z⁶ is CR. In certain instances of a fused tricyclic co-monomer offormula (I), one of Z⁵ and Z⁶ is CH. In certain instances of a fusedtricyclic co-monomer of formula (I), both of Z⁵ and Z⁶ are CH. In someembodiments of a fused tricyclic co-monomer of formula (I), Z⁵ is CR andZ⁶ is N. In certain cases of a fused tricyclic co-monomer of formula(I), Z⁵ is N and Z⁶ is CR.

In certain instances of formula (I), M¹, M² and M³ are independently aheterocyclic co-monomer wherein at least one of M¹, M² and M³ is athiophene containing co-monomer.

In some instances of formula (I), the multichromophore includes aconjugated segment having the structure of formula (Ia):

In certain embodiments of formula (I), M¹, M² and M³ are eachindependently a thiophene containing co-monomer. In certain instances offormula (I), a+c≦1. In certain instances of formula (I), d+f≦1. Incertain instances of formula (I), g+i≦1. In certain instances of formula(I), b+e+h≦1. In certain instances of formula (I), n+m+o≦1. In somecases, a+c≦1; d+f≦1;g+i≦1;b+e+h≦1; and n+m+o≦1. In certain instances, n≦1. In certain instances, m=0. In certain instances, o=0. In some cases,a+c=1. In certain cases, b=1 and e=1. In some cases, a+c=1. In certaincases, b+e+h=2. In some cases, d+f=1. In certain cases, b=1, e=1 andh=0. In some cases, g+i=1. In certain cases, b=1, e=0 and h=1.

In some embodiments of formula (I), the polymeric dye has the structureof formula (Ib):

In some instances of formula (Ib), each fused tricyclic co-monomer is ofthe same core structure, e.g., a fluorene, a carbazole, a silole or abridged bi-phenyl co-monomer. In some instances of formula (Ib), eachfused tricyclic co-monomer is independently selected from a fluorene, acarbazole, a silole and a bridged bi-phenyl co-monomer. In certaininstances of a fused tricyclic co-monomer of formula (Ib), one of Z⁵ andZ⁶ is CR. In some instances of a fused tricyclic co-monomer of formula(Ib), both of Z⁵ and Z⁶ are independently CR. In some embodiments of afused tricyclic co-monomer of formula (Ib), Z⁵ is CR and Z⁶ is N. Incertain cases of a fused tricyclic co-monomer of formula (Ib), Z⁵ is Nand Z⁶ is CR. In certain instances of a fused tricyclic co-monomer offormula (Ib), one of Z⁵ and Z⁶ is CH. In certain instances of a fusedtricyclic co-monomer of formula (Ib), both of Z⁵ and Z⁶ are CH. In someembodiments of a fused tricyclic co-monomer of formula (Ib), Z⁵ is CRand Z⁶ is N. In certain cases of a fused tricyclic co-monomer of formula(Ib), Z⁵ is N and Z⁶ is CR.

In some embodiments of formula (I) and (Ib), the polymeric dye has thestructure of formula (Ic):

In certain instances of formula (Ib)-(Ic), h, i and o are each 0. Incertain instances of formula (Ib)-(Ic), b and c are each 1. In certaininstances of formula (Ib)-(Ic), e and f are each 1. In certain instancesof formula (Ib)-(Ic), e, f and m are each 0. In certain instances offormula (Ib)-(Ic), h and i are each 1. In certain instances of formula(Ib)-(Ic), h, i and o are each 0; b and c are each 1; and e and f areeach 1. In certain cases, M² is linked to a signaling chromophore or achemoselective functional group. In certain instances of formula(Ib)-(Ic), e, f and m are each 0; b and c are each 1; and h and i areeach 1. In certain cases, Z¹ is a linked signaling chromophore. In somecases, Z¹ is a linked chemoselective functional group.

In some embodiments of formula (I), the polymeric dye has the structureof formula (II):

wherein u, v, w, x, y and z represent mol % values for each co-monomerin the multichromophore. In some instances of formula (IIa), each fusedtricyclic co-monomer is of the same core structure, e.g., a fluorene, acarbazole, a silole or a bridged bi-phenyl co-monomer. In some instancesof formula (IIa), each fused tricyclic co-monomer is independentlyselected from a fluorene, a carbazole, a silole and a bridged bi-phenylco-monomer. In certain instances of a fused tricyclic co-monomer offormula (IIa), one of Z⁵ and Z⁶ is CR. In some instances of a fusedtricyclic co-monomer of formula (IIa), both of Z⁵ and Z⁶ areindependently CR. In some embodiments of a fused tricyclic co-monomer offormula (IIa), Z⁵ is CR and Z⁶ is N. In certain cases of a fusedtricyclic co-monomer of formula (IIa), Z⁵ is N and Z⁶ is CR. In certaininstances of a fused tricyclic co-monomer of formula (IIa), one of Z⁵and Z⁶ is CH. In certain instances of a fused tricyclic co-monomer offormula (IIa), both of Z⁵ and Z⁶ are CH. In some embodiments of a fusedtricyclic co-monomer of formula (IIa), Z⁵ is CR and Z⁶ is N. In certaincases of a fused tricyclic co-monomer of formula (IIa), Z⁵ is N and Z⁶is CR. In certain instances, R is H, halogen, alkoxy, substitutedalkoxy, alkyl and substituted alkyl. In some instances, R is fluoro,methoxy lower alkyl or substituted lower alkyl.

In some embodiments, the polymeric dye has the structure of formula(IIb):

wherein u, v, w, x, y and z represent mol % values for each co-monomerin the multichromophore. It is understood that any of the polymeric dyesdescribed herein can also be described by a formula including mol %values for each of the co-monomers that are present, e.g., a formulacorresponding to formula (IIa)-(IIb). It is understood that any of thepolymeric dyes of formula (Ia) and (Ic) can be represented by formula(II). In some instances of formula (IIa)-(IIb), u+w+y constitutes 5% ormore by molarity (e.g., 5 mol %) of the multichromophore, such as 10% ormore, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more,40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% ormore, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more,or even more. In certain instances, z is 0. In some instances of formula(IIa)-(IIb), M¹, M² and M³ are each a thiophene-containing co-monomer.In some instances of formula (II), M¹ is a thiophene-containingco-monomer. In some instances of formula (IIa)-(IIb), M² is athiophene-containing co-monomer. In some instances of formula(IIa)-(IIb), M³ is a thiophene-containing co-monomer. In some instancesof formula (IIa)-(IIb), v+x+z constitutes 80% or less by molarity (e.g.,80 mol %) of the multichromophore, such as 75% or less, 70% or less, 65%or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% orless, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less,10% or less, or even less.

In some instances of formula (IIa)-(IIb), u is 5% or more, such as 10%or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% ormore, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more,65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% ormore, or even more. In some instances of formula (IIa)-(IIb), w is 5% ormore, such as 10% or more, 15% or more, 20% or more, 25% or more, 30% ormore, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more,60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% ormore, 90% or more, or even more. In some instances of formula(IIa)-(IIb), y is 1% or more, such as 2% or more, 5% or more, 10% ormore, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more,40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% ormore, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more,or even more. In some instances of formula (IIa)-(IIb), v is 80% orless, such as 75% or less, 70% or less, 65% or less, 60% or less, 55% orless, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less,25% or less, 20% or less, 15% or less, 10% or less, or even less. Insome instances of formula (IIa)-(IIb), x is 80% or less, such as 75% orless, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less,45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% orless, 15% or less, 10% or less, or even less. In some instances offormula (IIa)-(IIb), z is 80% or less, such as 75% or less, 70% or less,65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% orless, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less,10% or less, or even less.

In some embodiments of formula (I)-(IIb), the polymeric dye has thestructure of formula (IIIa):

In some instances of formula (IIIa), the fused tricyclic co-monomer isselected from a fluorene, a carbazole, a silole and a bridged bi-phenylco-monomer. In certain instances of formula (IIIa), one of Z⁵ and Z⁶ isCR. In some instances of formula (IIIa), both of Z⁵ and Z⁶ areindependently CR. In some embodiments of formula (IIIa), Z⁵ is CR and Z⁶is N. In certain cases of formula (IIIa), Z⁵ is N and Z⁶ is CR. Incertain instances of formula (IIIa), one of Z⁵ and Z⁶ is CH. In certaininstances of formula (IIIa), both of Z⁵ and Z⁶ are CH. In someembodiments of formula (IIIa), Z⁵ is CR and Z⁶ is N. In certain cases offormula (IIIa), Z⁵ is N and Z⁶ is CR. In certain instances, R is H,halogen, alkoxy, substituted alkoxy, alkyl and substituted alkyl. Insome instances, R is fluoro, methoxy lower alkyl or substituted loweralkyl.

In some embodiments of formula (I)-(IIIa), the polymeric dye has thestructure of formula (IIIb):

wherein n is an integer from 1 to 100,000 and M¹ is athiophene-containing co-monomer. In certain embodiments of formula(III)-(IIIb), Y¹ is C(R³)₂. In certain embodiments of formula(III)-(IIIb), Y¹ is —C(R³)₂C(R³)₂—. In some cases of formula(IIIa)-(IIIb), Y² is —CHR³CHR³—. In certain instances of mula(IIIa)-(IIIb), Y¹ is NR³. In certain embodiments of formula(III)-(IIIb), Y¹ is Si(R³)₂. In some instances, each R³ is independentlyselected from H, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy,substituted alkoxy, amido, substituted amido, substituted sulfonamidoand a WSG. In certain instances, at least one R³ is a WSG. In certaincases, both R³ groups are independently a WSG.

In some embodiments of formula (I)-(IIa), the polymeric dye has thestructure of formula (IVa):

wherein: M¹ and M² are each independently a thiophene containingco-monomer; each n and m are independently an integer from 1 to 10,000;and p is an integer from 1 to 100,000. In some instances of formula(IVa), each fused tricyclic co-monomer is of the same core structure,e.g., a fluorene, a carbazole, a silole or a bridged bi-phenylco-monomer. In some instances of formula (IVa), each fused tricyclicco-monomer is independently selected from a fluorene, a carbazole, asilole and a bridged bi-phenyl co-monomer. In some instances of formula(IVa), the fused tricyclic co-monomer is selected from a fluorene, acarbazole, a silole and a bridged bi-phenyl co-monomer. In certaininstances of formula (IVa), one of Z⁵ and Z⁶ is CR. In some instances offormula (IVa), both of Z⁵ and Z⁶ are independently CR. In someembodiments of formula (IVa), Z⁵ is CR and Z⁶ is N. In certain cases offormula (IVa), Z⁵ is N and Z⁶ is CR. In certain instances of formula(IVa), one of Z⁵ and Z⁶ is CH. In certain instances of formula (IVa),both of Z⁵ and Z⁶ are CH. In some embodiments of formula (IVa), Z⁵ is CRand Z⁶ is N. In certain cases of formula (IVa), Z⁵ is N and Z⁶ is CR. Incertain instances, R is H, halogen, alkoxy, substituted alkoxy, alkyland substituted alkyl. In some instances, R is fluoro, methoxy loweralkyl or substituted lower alkyl.

In some embodiments of formula (I)-(IVa), the polymeric dye has thestructure of formula (IVb):

wherein: M¹ and M² are each independently a thiophene containingco-monomer; each n and m are independently an integer from 1 to 10,000;and p is an integer from 1 to 100,000. In certain instances of formula(IVa)-(IVb), Y¹ is NR³; Y² is C(R³)₂; and M¹ and M² are the samethiophene containing co-monomer. In certain instances of formula(IVa)-(IVb), Y¹ is C(R³)₂; Y² is NR³; and M¹ and M² are the samethiophene containing co-monomer. In certain instances of formula(IVa)-(IVb), Y¹ and Y² are each independently C(R³)₂; and M¹ and M² arethe same thiophene containing co-monomer. In certain instances offormula (IVa)-(IVb), Y¹ is NR³; Y² is C(R³)₂; and M¹ and M² aredifferent thiophene containing co-monomers. In certain instances offormula (IVa)-(IVb), Y¹ is C(R³)₂; Y² is NR³; and M¹ and M² aredifferent thiophene containing co-monomers. In certain instances offormula (IVa)-(IVb), Y¹ and Y² are each independently C(R³)₂; and M¹ andM² are different thiophene containing co-monomers. In certain instancesof formula (IVa)-(IVb), Y¹ and Y² are each independently Si(R³)₂. Incertain instances of formula (IVa)-(IVb), Y¹ is Si(R³)₂. In certaininstances of formula (IVa)-(IVb), Y² is Si(R³)₂. In some cases offormula (IVa)-(IVb), Y¹ is —C(R³)₂C(R³)₂—. In some cases of formula(IVa)-(IVb), Y¹ is —CHR³CHR³. In some cases of formula (IVa)-(IVb), Y²is —C(R³)₂C(R³)₂—. In some cases of formula (IVa)-(IVb), Y² is—CHR³CHR³. In some instances, each R³ is independently selected from H,alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, acyl, substituted acyl, alkoxy, substitutedalkoxy, amido, substituted amido and a WSG. In certain instances, atleast one R³ is a WSG. In certain cases, both R³ groups areindependently a WSG.

In certain embodiments of formula (I)-(IIIb), the polymeric dye has thestructure of formula (Va):

wherein: M¹ and M³ are each independently a thiophene containingco-monomer; each n and each o are independently an integer from 1 to10,000; and p is an integer from 1 to 100,000. In some instances offormula (Va), each fused tricyclic co-monomer is of the same corestructure, e.g., a fluorene, a carbazole, a silole or a bridgedbi-phenyl co-monomer. In some instances of formula (Va), each fusedtricyclic co-monomer is independently selected from a fluorene, acarbazole, a silole and a bridged bi-phenyl co-monomer. In someinstances of formula (Va), the fused tricyclic co-monomer is selectedfrom a fluorene, a carbazole, a silole and a bridged bi-phenylco-monomer. In certain instances of formula (Va), one of Z⁵ and Z⁶ isCR. In some instances of formula (Va), both of Z⁵ and Z⁶ areindependently CR. In some embodiments of formula (Va), Z⁵ is CR and Z⁶is N. In certain cases of formula (Va), Z⁵ is N and Z⁶ is CR. In certaininstances of formula (Va), one of Z⁵ and Z⁶ is CH. In certain instancesof formula (Va), both of Z⁵ and Z⁶ are CH. In some embodiments offormula (Va), Z⁵ is CR and Z⁶ is N. In certain cases of formula (Va), Z⁵is N and Z⁶ is CR. In certain instances, R is H, halogen, alkoxy,substituted alkoxy, alkyl and substituted alkyl. In some instances, R isfluoro, methoxy lower alkyl or substituted lower alkyl.

In certain embodiments of formula (I)-(IIb), the polymeric dye has thestructure of formula (Vb):

wherein: M¹ and M³ are each independently a thiophene containingco-monomer; each n and each o are independently an integer from 1 to10,000; and p is an integer from 1 to 100,000. In some instances offormula (Va)-(Vb), Y³—Z¹ is C(R³)(T¹—Z¹), wherein Z¹ is a chemoselectivefunctional group or a linked signaling chromophore and T¹ is a linker.In some cases of formula (Va)-(Vb), Y³—Z¹ is N-T¹-Z¹, wherein Z¹ is achemoselective functional group or a linked signaling chromophore and T¹is a linker. In some instances of formula (Va)-(Vb), Y³—Z¹ isSi(R³)(T¹—Z¹), wherein Z¹ is a chemoselective functional group or alinked signaling chromophore and T¹ is a linker. In certain instances,Z¹ is a chemoselective functional group. In some instances, Z¹ is alinked signaling chromophore. In some embodiments of formula (Va)-(Vb),Y¹ is NR³; and M¹ and M³ are the same thiophene containing co-monomer.In some embodiments of formula (Va)-(Vb), Y¹ is C(R³)₂; and M¹ and M³are the same thiophene containing co-monomer. In some instances offormula (Va)-(Vb), Y¹ is NR³; and M¹ and M³ are different thiophenecontaining co-monomers. In some embodiments of formula (Va)-(Vb), Y¹ isC(R³)₂; and M¹ and M³ are different thiophene containing co-monomers. Insome cases of formula (Va)-(Vb), Y¹ is —C(R³)₂C(R³)₂—. In some cases offormula (Va)-(Vb), Y¹ is —CHR³CHR³—. In some embodiments of formula(Va)-(Vb), Y¹ is Si(R³)₂. In some embodiments of formula (Va)-(Vb), M¹and M³ are the same thiophene containing co-monomer. In some embodimentsof formula (Va)-(Vb), M¹ and M³ are different thiophene containingco-monomers.

In certain embodiments of formula (1)-(11), the polymeric dye has thestructure of formula (VIa):

wherein: at least one of M¹ and M² is a thiophene containing co-monomer;each n and each o are independently an integer from 1 to 10,000; Z¹ is alinked chemoselective functional group or a linked signalingchromophore; and p is an integer from 1 to 100,000. In some instances offormula (VIa), each fused tricyclic co-monomer is of the same corestructure, e.g., a fluorene, a carbazole, a silole or a bridgedbi-phenyl co-monomer. In some instances of formula (VIa), each fusedtricyclic co-monomer is independently selected from a fluorene, acarbazole, a silole and a bridged bi-phenyl co-monomer. In certaininstances of a fused tricyclic co-monomer of formula (VIa), one of Z⁵and Z⁶ is CR. In some instances of a fused tricyclic co-monomer offormula (VIa), both of Z⁵ and Z⁶ are independently CR. In someembodiments of a fused tricyclic co-monomer of formula (VIa), Z⁵ is CRand Z⁶ is N. In certain cases of a fused tricyclic co-monomer of formula(VIa), Z⁵ is N and Z⁶ is CR. In certain instances of a fused tricyclicco-monomer of formula (VIa), one of Z⁵ and Z⁶ is CH. In certaininstances of a fused tricyclic co-monomer of formula (VIa), both of Z⁵and Z⁶ are CH. In some embodiments of a fused tricyclic co-monomer offormula (VIa), Z⁵ is CR and Z⁶ is N. In certain cases of a fusedtricyclic co-monomer of formula (VIa), Z⁵ is N and Z⁶ is CR. In certaininstances, R is H, halogen, alkoxy, substituted alkoxy, alkyl andsubstituted alkyl. In some instances, R is fluoro, methoxy lower alkylor substituted lower alkyl.

In certain embodiments of formula (I)-(VIa), the polymeric dye has thestructure of formula (VIb):

wherein: at least one of M¹ and M² is a thiophene containing co-monomer;each n and each o are independently an integer from 1 to 10,000; Z¹ is alinked chemoselective functional group or a linked signalingchromophore; and p is an integer from 1 to 100,000. In some cases offormula (VIa)-(VIb), M¹ and M² are each independently a thiophenecontaining co-monomer. In some instances of formula (VIa)-(VIb), Z¹ is alinked chemoselective functional group. In some instances of formula(VIa)-(VIb), Z¹ is a linked signaling chromophore. In some embodimentsof formula (VIa)-(VIb), Y¹ is NR³. In some embodiments of formula(VIa)-(VIb), Y¹ is C(R³)₂. In some embodiments of formula (VIa)-(VIb),Y² is NR³. In some embodiments of formula (VIa)-(VIb), Y² is C(R³)₂. Incertain instances of formula (VIa)-(VIb), Y¹ and Y² are eachindependently Si(R³)₂. In certain instances of formula (VIa)-(VIb), Y¹is Si(R³)₂. In certain instances of formula (VIa)-(VIb), Y² is Si(R³)₂.In some cases of formula (VIa)-(VIb), Y¹ is —C(R³)₂C(R³)₂—. In somecases of formula (VIa)-(VIb), Y¹ is —CHR³CHR³. In some cases of formula(VIa)-(VIb), Y² is —C(R³)₂C(R³)₂—. In some cases of formula (VIa)-(VIb),Y² is —CHR³CHR³. In some instances of formula (VIa)-(VIb), M¹ and M²have the same underlying unsubstituted thiophene containing co-monomer,except M² includes a linked Z¹, and M¹ is optionally substituted with aR³ group. In some instances of formula (VIa)-(VIb), M¹ and M² aredifferent thiophene containing co-monomers. In some instances, each R³is independently selected from H, alkyl, substituted alkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, acyl, substitutedacyl, alkoxy, substituted alkoxy, amido, substituted amido and a WSG. Incertain instances, at least one R³ is a WSG. In certain cases, both R³groups are independently a WSG.

Thiophene-Containing Co-Monomers

Aspects of the present disclosure include water solvated polymeric dyes(e.g., as described herein, such as a polymeric dye of one of formula(I)-(VIb)) including a thiophene-containing co-monomer. The term“thiophene-containing co-monomer” refers to a heterocyclic group orsubstituted heterocyclic group that includes a thiophene ring and iscapable of conjugation with a pi-conjugated polymer backbone viaadjacent co-monomers. A thiophene that is a 5-membered aromaticheterocyclic ring having the formula C₄H₄S, which can be substitutedwith any convenient substituents (e.g., at the 2, 3, 4 and/or 5positions and/or linked or fused to any convenient aryl or heteroarylring systems (e.g., fused to an additional ring at the 2,3-, 3,4- and/or4,5- positions). The co-monomer is divalent and as such is substitutedat two sites of the ring system for covalent attachment to theunsaturated backbone of a conjugated polymer. In some cases, thethiophene ring of the co-monomer is connected to the conjugated polymerbackbone via positions 2 and/or 5 of the thiophene ring.

Any convenient thiophene-containing heterocyclic groups can be adaptedfor use as a thiophene-containing co-monomer in the subject conjugatedpolymers. Thiophene-containing heterocyclic groups of interest include,but are not limited to, thiophene, 1-benzothiophene, 2-benzothiophene,dibenzothiophene, and polythiophene such as fused dithiophene or fusedtrithiophene, which groups can be connected to the conjugated polymervia any two convenient positions of the group and can be optionallyfurther substituted, e.g., with a WSG. In some cases, thethiophene-containing co-monomer is monocyclic (e.g., a substitutedthiophene). In some cases, the thiophene-containing co-monomer comprisesa fused thiophene ring, such as a 1-benzothiophene, 2-benzothiophene andsubstituted versions thereof. In some cases, the thiophene-containingco-monomer comprises an aryl or heteroaryl ring system as a substituentthat is not directly connected to the conjugated polymer backbone.

In some embodiments of formulae (I)-(VIb), the thiophene-containingco-monomer(s) (M¹-M³) are selected from formula (VII), (VIII) and (IX):

wherein:

A1 and A2 are each a fused monocyclic or bicyclic group

each R³ is independently selected from H, amino, substituted amino,halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido,substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy, WSGand -T²-Z², wherein Z² is a chemoselective functional group or a linkedacceptor chromophore, and T² is a linker;

n is 0, 1, 2, 3 or 4; and

each * is a site for covalent attachment to the unsaturated backbone ofa conjugated polymer. In some instances of formulae (I)-(VIb), athiophene-containing co-monomer (e.g., M¹, M² and/or M³) is described byformula (VII). In some instances of formulae (I)-(VIb), athiophene-containing co-monomer (e.g., M¹, M² and/or M³) is described byformula (VIII). In some instances of formulae (I)-(VIb), thethiophene-containing co-monomer (e.g., M¹, M² and/or M³) is described byformula (IX).

Fused monocyclic or bicyclic groups of interest include fusedcarbocyclic and heterocyclic groups having one or two 5, 6 and/or7-membered rings which when fused with the thiophene ring form a fusedtwo or three-ring system. The fused carbocyclic and heterocylic ringscan be saturated or unsaturated. In certain instances of formula(VIII)-(IX), A1 and A2 are a fused monocyclic group, such as a fusedbenzo ring, a fused pyridyl ring, a fused benzothiophene ring system ora fused 7-membered heterocycle. In some instances of formula(VIII)-(IX), A1 and A2 are selected from a fused monocyclic heterocycle,a fused monocyclic heteroaryl and a fused bicyclic heteroaryl.

In some embodiments of formulae (VII)-(IX), the thiophene-containingco-monomer(s) are selected from formulae (X)-(XVII):

wherein:

X¹ is CR³, NR³, O or S;

each X² is independently N, O or S, wherein when X² is N, the adjacent

is a double bond and when X² is O or S, the adjacent

is a single bond;

each X³ is independently O, S or CH;

R¹ and R² together form a 5- or 6-membered fused aryl or heteroaryl ringwhich is optionally substituted with one or more R³ groups; and

Y⁴ is NR³, C(R³)₂ or Si(R³)₂.

In certain embodiments of formula (XV), X¹ is CR³. In certainembodiments of formula (XV), X¹ is CH. In certain cases of formula (XV),X¹ is NR³. In certain instances of formula (XV), X¹ is O. In someembodiments of formula (XV), X¹ is S. In certain instances of formula(XV), R³ is a WSG and X¹ is O or S.

In certain embodiments of formula (XII)-(XIII), each X² is N. In certainembodiments of formula (XIII)-(XIV), each X² is O. In certainembodiments of formula (XIII)-(XIV), each X² is S. In certainembodiments of formula (XII), each X³ is O. In certain embodiments offormula (XII), each X³ is S. In certain embodiments of formula (XII),each X³ is CH.

In some embodiments of the polymeric dye, the thiophene-containingco-monomers (e.g., co-monomers M¹-M³) are independently selected fromformulae (XVIII) and (XIX):

wherein:

R¹ and R² together form a 5- or 6-membered fused aryl or heteroaryl ringwhich is optionally substituted with one or more R³ groups; and

each R³ is independently selected from H, amino, substituted amino,halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido,substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy and-T²-Z², wherein Z² is a chemoselective functional group or a linkedacceptor chromophore, and T² is a linker.

In some embodiments of formula (I)-(XVII), a thiophene-containingco-monomer (e.g., M¹, M² and/M³) is described by one of the followingstructures (a) to (q):

wherein:

each R⁴ is independently selected from H, amino, substituted amino,halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido,substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy, WSGand -T²-Z², wherein Z² is a chemoselective functional group or a linkedsignaling chromophore, and T² is a linker; and

each s is 0 or an integer from 1 to 50. In certain instances, each s isindependently 1 to 20, such as 3 to 20, 3 to 15, 3 to 12, or 6 to 12. Incertain cases, each s is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11or 12. In some cases, each s is 3. In some cases, each s is 4. In somecases, each s is 5. In some cases, each s is 6. In some cases, each s is7. In some cases, each s is 8. In some cases, each s is 9. In somecases, each s is 10. In some cases, each s is 11. In some instances, atleast one R⁴ is -T²-Z². In some embodiments, T² is an alkyl linker, suchas a C1-C6alkyl linker. In some embodiments, T² is a substituted alkyllinker. In some embodiments, T² is an alkoxy linker (e.g., —O-alkyl-).In some embodiments, T² is a substituted alkoxy. In some instances, Z²is a chemoselective functional group. In some instances, Z² is a linkedsignaling chromophore. In some instances, each R⁴ is a WSG. In someinstances, each R⁴ is a substituted alkyl group. In some instances, eachR⁴ is an alkoxy or a substituted alkoxy group.

The thiophene-containing co-monomers (e.g., co-monomers M¹-M³) can beany convenient fused tricyclic co-monomer. Tricyclic co-monomers ofinterest include, but are not limited to, those co-monomers includingtwo thiophene rings connected via a fused central 5- or 6-membered arylor heteroaryl ring. The fused central ring can be a pyrrole, a benzo ora cyclopentadiene. In some embodiments of the polymeric dye, thethiophene-containing co-monomers (e.g., co-monomers M¹-M³) areindependently selected from formulae (e)-(i):

wherein: each R⁴ is independently selected from H, amino, substitutedamino, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido,substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy and-T²-Z², wherein Z² is a chemoselective functional group or a linkedacceptor chromophore, and T² is a linker; and each s is 0 or an integerfrom 1 to 50 (e.g., s is 1 to 20, etc., such as s is 11).

In some embodiments of formula (I)-(XVI), at least onethiophene-containing co-monomer is described by structure (a). In someembodiments of formula (I)-(XVI), at least one thiophene-containingco-monomer is described by structure (b). In some embodiments of formula(I)-(XVI), at least one thiophene-containing co-monomer is described bystructure (c). In some embodiments of formula (I)-(XVI), at least onethiophene-containing co-monomer is described by structure (d). In someembodiments of formula (I)-(XVI), at least one thiophene-containingco-monomer is described by structure (e). In some embodiments of formula(I)-(XVI), at least one thiophene-containing co-monomer is described bystructure (f). In some embodiments of formula (I)-(XVI), at least onethiophene-containing co-monomer is described by structure (g). In someembodiments of formula (I)-(XVI), at least one thiophene-containingco-monomer is described by structure (h). In some embodiments of formula(I)-(XVI), at least one thiophene-containing co-monomer is described bystructure (i). In some embodiments of formula (I)-(XVI), at least onethiophene-containing co-monomer is described by structure (j). In someembodiments of formula (I)-(XVI), at least one thiophene-containingco-monomer is described by structure (k). In some embodiments of formula(I)-(XVI), at least one thiophene-containing co-monomer is described bystructure (I). In some embodiments of formula (I)-(XVI), at least onethiophene-containing co-monomer is described by structure (m). In someembodiments of formula (I)-(XVI), at least one thiophene-containingco-monomer is described by structure (n). In some embodiments of formula(I)-(XVI), at least one thiophene-containing co-monomer is described bystructure (o). In some embodiments of formula (I)-(XVI), at least onethiophene-containing co-monomer is described by structure (p). In someembodiments of formula (I)-(XVI), at least one thiophene-containingco-monomer is described by structure (q).

In some embodiments of formula (I)-(XVII) and (a)-(q), athiophene-containing co-monomer (e.g., M¹, M² and/M³) is described byone of the following structures (ba) to (bu):

In some instances, at least one thiophene-containing co-monomer (e.g.,M¹, M² and/M³) has structure (ba). In some instances, at least onethiophene-containing co-monomer (e.g., M¹, M² and/M³) has structure(bb). In some instances, at least one thiophene-containing co-monomer(e.g., M¹, M² and/M³) has structure (bc). In some instances, at leastone thiophene-containing co-monomer (e.g., M¹, M² and/M³) has structure(bd). In some instances, at least one thiophene-containing co-monomer(e.g., M¹, M² and/M³) has structure (be). In some instances, at leastone thiophene-containing co-monomer (e.g., M¹, M² and/M³) has structure(bf). In some instances, at least one thiophene-containing co-monomer(e.g., M¹, M² and/M³) has structure (bg). In some instances, at leastone thiophene-containing co-monomer (e.g., M¹, M² and/M³) has structure(bh). In some instances, at least one thiophene-containing co-monomer(e.g., M¹, M² and/M³) has structure (bi). In some instances, at leastone thiophene-containing co-monomer (e.g., M¹, M² and/M³) has structure(bj). In some instances, at least one thiophene-containing co-monomer(e.g., M¹, M² and/M³) has structure (bk). In some instances, at leastone thiophene-containing co-monomer (e.g., M¹, M² and/M³) has structure(bl). In some instances, at least one thiophene-containing co-monomer(e.g., M¹, M² and/M³) has structure (bm). In some instances, at leastone thiophene-containing co-monomer (e.g., M¹, M² and/M³) has structure(bn). In some instances, at least one thiophene-containing co-monomer(e.g., M¹, M² and/M³) has structure (bo). In some instances, at leastone thiophene-containing co-monomer (e.g., M¹, M² and/M³) has structure(bp). In some instances, at least one thiophene-containing co-monomer(e.g., M¹, M² and/M³) has structure (bq). In some instances, at leastone thiophene-containing co-monomer (e.g., M¹, M² and/M³) has structure(br). In some instances, at least one thiophene-containing co-monomer(e.g., M¹, M² and/M³) has structure (bs). In some instances, at leastone thiophene-containing co-monomer (e.g., M¹, M² and/M³) has structure(bt). In some instances, at least one thiophene-containing co-monomer(e.g., M¹, M² and/M³) has structure (bu).

In some embodiments of formulae (I)-(VI), the polymeric dye includes aconjugated segment having one of the following structures:

where n is 1 to 100,000 and each R¹¹, R¹² and R¹³ is independently asubstituted alkyl, a substituted aralkyl, or a WSG.

In some embodiments of formulae (I), (Ib), (II) and (V), the polymericdye includes a conjugated segment having one of the followingstructures:

where n and o are independently 1 to 10,000 and each R¹¹, R¹² and R¹³ isindependently a substituted alkyl, a substituted aralkyl, or a WSG. Insome embodiments of formulae (I), (Ib), (II) and (VI), the polymeric dyeincludes a conjugated segment having the following structure:

where n and m are independently 1 to 10,000 and each R¹¹ isindependently a substituted alkyl, a substituted aralkyl, or a WSG. Insome embodiments of formulae (I), (Ib), (II) and (VI), the polymeric dyeincludes a conjugated segment having one of the following structures:

where n and m are independently 1 to 10,000 and each R¹¹, R¹² and R¹³ isindependently a substituted alkyl, a substituted aralkyl, or a WSG.

In certain instances, each R¹¹, R¹² and R¹³ of the conjugated segment isindependently selected from the following substituents:

where each s is an integer from 1 to 50. In certain instances, each s isindependently 1 to 20, such as 3 to 20, 3 to 15, 3 to 12, or 6 to 12. Incertain cases, each s is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11or 12. In some cases, each s is 3. In some cases, each s is 4. In somecases, each s is 5. In some cases, each s is 6. In some cases, each s is7. In some cases, each s is 8. In some cases, each s is 9. In somecases, each s is 10. In some cases, each s is 11. In certain instances,each R¹¹ is substituent (yy), wherein s is 3 to 20 (e.g., 11). Incertain instances, each R¹² is substituent (zz), wherein s is 3 to 20(e.g., 11). In certain instances, each R¹³ is substituent (xx), whereins is 3 to 20 (e.g., 11). In certain embodiments of any one of theconjugated segment structures, a terminal of the polymeric dye includesthe following linker.

Water Solubilizing Substituents

The subject polymeric dyes may be water solvated. Any convenient watersolubilizing groups (WSG's) may be included in the multichromophoresdescribed herein (e.g., multichromophores of formulae (I)-(VIb),co-monomers of formulae (VII)-(XVII) and (a)-(q)) to provide forincreased water-solubility. While the increase in solubility may vary,in some instances the increase (as compared to the compound without theWSG(s)) is 2 fold or more, e.g., 5 fold, 10 fold, 25 fold, 50 fold, 100fold or more. As used herein, the terms “water solubilizing group”,“water soluble group” and WSG are used interchangeably and refer to agroup or substituent that is well solvated in aqueous environments e.g.,under physiological conditions, and that imparts improved watersolubility upon the molecules to which it is attached. In someembodiments, a WSG increases the solubility of the multichromophore in apredominantly aqueous solution, as compared to a controlmultichromophore which lacks the WSG. In some instances, the WSGs of themultichromophore are non-ionic side groups capable of impartingsolubility in water in excess of 10 mg/mL. The water solubilizing groupsmay be any convenient hydrophilic group that is well solvated in aqueousenvironments. In some cases, the hydrophilic water solubilizing group ischarged, e.g., positively or negatively charged. In certain cases, thehydrophilic water solubilizing group is a neutral hydrophilic group. Insome embodiments, the WSG is a hydrophilic polymer, e.g., a polyethyleneglycol, a modified PEG, a peptide sequence, a peptoid, a carbohydrate,an oxazoline, a polyol, a dendron, a dendritic polyglycerol, acellulose, a chitosan, or a derivative thereof. Water solubilizinggroups of interest include, but are not limited to, carboxylate,phosphonate, phosphate, sulfonate, sulfate, sulfinate, sulfonium, ester,polyethylene glycols (PEG) and modified PEGs, hydroxyl, amine, aminoacid, ammonium, guanidinium, pyridinium, polyamine and sulfonium,polyalcohols, straight chain or cyclic saccharides, primary, secondary,tertiary, or quaternary amines and polyamines, phosphonate groups,phosphinate groups, ascorbate groups, glycols, including, polyethers,—COOM′, —SO₃M′, —PO₃M′, —NR₃ ⁺, Y′, (CH₂CH₂O)_(p)R and mixtures thereof,where Y′ can be any halogen, sulfate, sulfonate, or oxygen containinganion, p can be 1 to 500, each R can be independently H or an alkyl(such as methyl) and M′ can be a cationic counterion or hydrogen,—(CH₂CH₂O)_(yy)CH₂CH₂XR^(yy), —(CH₂CH₂O)_(yy)CH₂CH₂X—,—X(CH₂CH₂O)_(yy)CH₂CH₂—, glycol, and polyethylene glycol, wherein yy isselected from 1 to 1000, X is selected from O, S, and NR^(ZZ), andR^(ZZ) and R^(YY) are independently selected from H and C₁₋₃alkyl. Insome cases, a WSG is (CH₂)_(x)(OCH₂CH₂)_(y)OCH₃ where each x isindependently an integer from 0-20, each y is independently an integerfrom 0 to 50. In some cases, the water solubilizing group includes anon-ionic polymer (e.g., a PEG polymer) substituted at the terminal withan ionic group (e.g., a sulfonate).

In some embodiments of formulae, the co-monomer includes a substituentselected from (CH₂)_(x)(OCH₂CH₂)_(y)OCH₃ where each x is independentlyan integer from 0-20, each y is independently an integer from 0 to 50;and a benzyl optionally substituted with one or more halogen, hydroxyl,C₁-C₁₂alkoxy, or (OCH₂CH₂)_(z)OCH₃ where each z is independently aninteger from 0 to 50. In some instances, the substituent is(CH₂)₃(OCH₂CH₂)₁₁OCH₃. In some embodiments, one or more of thesubstituents is a benzyl substituted with at least one WSG groups (e.g.,one or two WSG groups) selected from (CH₂)_(x)(OCH₂CH₂)_(y)OCH₃ whereeach x is independently an integer from 0-20 and each y is independentlyan integer from 0 to 50.

Multiple WSGs may be included at a single location in the subjectmultichromophores via a branching linker. In certain embodiments, thebranching linker is an aralkyl substituent, further di-substituted withwater solubilizing groups. As such, in some cases, the branching linkergroup is a substituent of the multichromophore that connects themultichromophore to two or more water solubilizing groups. In certainembodiments, the branching linker is an amino acid, e.g., a lysine aminoacid that is connected to three groups via the amino and carboxylic acidgroups. In some cases, the incorporation of multiple WSGs via branchinglinkers imparts a desirable solubility on the multichromophore. In someinstances, the WSG is a non-ionic sidechain group capable of impartingsolubility in water in excess of 10 mg/mL.

In some embodiments, the multichromophore includes substituent(s)selected from the group consisting of, an alkyl, an aralkyl and aheterocyclic group, each group further substituted with a include watersolubilizing groups hydrophilic polymer group, such as a polyethylglycol(PEG) (e.g., a PEG group of 2-20 units).

In certain instances of any one of formulae (I)-(XVII) and (a)-(q), oneor more of the co-monomers may be substituted with a group (e.g., R³ andR⁴ substituent groups of the depicted formulae) that is independentlyselected from H, substituted aryl, alkyl and one of the followingstructures:

wherein: each T⁵ is independently an optional linker; and each s is aninteger from 1 to 50. In certain instances, each s is independently 1 to20, such as 3 to 20, 3 to 15, 3 to 12, or 6 to 12. In certain cases,each s is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In somecases, each s is 3. In some cases, each s is 4. In some cases, each s is5. In some cases, each s is 6. In some cases, each s is 7. In somecases, each s is 8. In some cases, each s is 9. In some cases, each s is10. In some cases, each s is 11. In some cases, each s is 12. In somecases, each s is 14. In some cases, each s is 16. In some embodiments,T⁵ is an alkyl linker, such as a C1-C6alkyl linker. In some embodiments,T⁵ is a substituted alkyl linker. In some embodiments, T⁵ is an alkoxylinker (e.g., —O-alkyl-). In some embodiments, T⁵ is a substitutedalkoxy. It is understood that hydroxy-terminated PEG chains instead ofmethoxy-terminated PEG chains may be utilized in any of the WSG groupsdescribed herein.

In some cases, a WSG is a dendron selected from one of the followingstructures:

In some cases, a WSG is a polyol selected from one of the followingstructures:

In some cases, a WSG is an oxazoline of the following structure:

In some cases, a WSG is a peptiod selected from one of the followingstructures:

Polymeric Tandem Dyes

The water soluble light harvesting multichromophore can itself befluorescent and capable of transferring energy to a linked acceptorsignaling chromophore. As such, the subject polymeric tandem dyesfurther include a covalently linked acceptor signaling chromophore inenergy-receiving proximity to the donor water solvated light harvestingmultichromophore. As such, excitation of the multichromophore donorleads to energy transfer to and emission from the covalently attachedacceptor signaling chromophore. The number of signaling chromophoreacceptor units that are linked to the donor water solvated lightharvesting multichromophore may vary, where in some instances the numberranges from 1 mol % to 50 mol %, such as from 5 mol % to 25 mol % orfrom 10 mol % to 25 mol %.

Mechanisms for energy transfer from the fluorescent water solvated lightharvesting multichromophore donor to the linked acceptor signalingchromophroe include, for example, resonant energy transfer (e.g.,Förster (or fluorescence) resonance energy transfer, FRET), quantumcharge exchange (Dexter energy transfer) and the like. In someinstances, these energy transfer mechanisms are relatively short range;that is, close proximity of the light harvesting multichromophore systemto the acceptor provides for efficient energy transfer. In someinstances, under conditions for efficient energy transfer, amplificationof the emission from the acceptor occurs where the emission from theluminescent signaling chromophore is more intense when the incidentlight (the “pump light”) is at a wavelength which is absorbed by thelight harvesting multichromophore than when the luminescent signalingchromophore is directly excited by the pump light.

By “efficient” energy transfer is meant 10% or more, such as 20% or moreor 30% or more, of the energy harvested by the donor is transferred tothe acceptor. By “amplification” is meant that the signal from thesignaling chromophore is 1.5× or greater when excited by energy transferfrom the donor light harvesting multichromophore as compared to directexcitation with incident light of an equivalent intensity. The signalmay be measured using any convenient method. In some cases, the 1.5× orgreater signal refers to an intensity of emitted light. In certaincases, the 1.5× or greater signal refers to an increased signal to noiseratio. In certain embodiments of the polymeric tandem dye, the signalingchromophore emission is 1.5 fold greater or more when excited by themultichromophore as compared to direct excitation of the signalingchromophore with incident light, such as 2-fold or greater, 3-fold orgreater, 4-fold or greater, 5-fold or greater, 6-fold or greater, 8-foldor greater, 10-fold or greater, 20-fold or greater, 50-fold or greater,100-fold or greater, or even greater as compared to direct excitation ofthe signaling chromophore with incident light.

The linked luminescent signaling chromophore emission of the polymerictandem dye can have a quantum yield of 0.03 or more, such as a quantumyield of 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 ormore, 0.09 or more, 0.1 or more, 0.15 or more, 0.2 or more, 0.3 or moreor even more. In some instances, the polymeric tandem dye has anextinction coefficient of 5×10⁵ cm⁻¹M⁻¹ or more, such as 6×10⁵ cm⁻¹M⁻¹or more, 7×10⁵ cm⁻¹M⁻¹ or more, 8×10⁵ cm⁻¹M⁻¹ or more, 9×10⁵ cm⁻¹M⁻¹ ormore, such as 1×10⁶ cm⁻¹M⁻¹ or more, 1.5×10⁶ cm⁻¹M⁻¹ or more, 2×10⁶cm⁻¹M⁻¹ or more, 2.5×10 ⁶ cm⁻¹M⁻¹ or more, 3×10⁶ cm⁻¹M⁻¹ or more, 4×10⁶cm⁻¹M⁻¹ or more, 5×10⁶ cm ⁻¹M⁻¹ or more, 6×10⁶ cm⁻¹M⁻¹ or more, 7×10⁶cm⁻¹M⁻¹ or more, or 8×10⁶ cm⁻¹M⁻¹ or more. In some embodiments, thepolymeric tandem dye has a molar extinction coefficient of 5×10⁵ M⁻¹cm⁻¹ or more. In certain embodiments, the polymeric tandem dye has amolar extinction coefficient of 1×10⁶ M⁻¹ cm⁻¹ or more.

The subject polymeric tandem dyes provide for fluorescence emissionsfrom luminescent signaling chromophore dyes that are brighter than theemissions which are possible from such luminescent dyes in isolation.The linked luminescent signaling chromophore emission of the polymerictandem dye can have a brightness of 50 mM⁻¹ cm⁻¹ or more, such as 60mM⁻¹ cm⁻¹ or more, 70 mM⁻¹ cm⁻¹ or more, 80 mM⁻¹ cm⁻¹ or more, 90 mM⁻¹cm⁻¹ or more, 100 mM⁻¹ cm⁻¹ or more, 150 mM⁻¹ cm⁻¹ or more, 200 mM⁻¹cm⁻¹ or more, 250 mM⁻¹ cm ⁻¹ or more, 300 mM⁻¹ cm ⁻¹ or more, or evenmore. In certain instances, the linked signaling chromophore emission ofthe polymeric tandem dye has a brightness that is at least 5-foldgreater than the brightness of a directly excited luminescent dye, suchas at least 10-fold greater, at least 20-fold greater, at least 30-foldgreater, at least 50-fold greater, at least 100-fold greater, at least300-fold greater, or even greater than the brightness of a directlyexcited luminescent dye.

In some embodiments, a polymeric tandem dye is of one of formula(I)-(VI), where a linked signaling chromophore (e.g., as describedherein) is included as a substituent of a co-monomer. In some instancesof formula (I)-(VI), the polymeric tandem dye includes one or moreco-monomers as described by formula (VII)-(XVII), (a)-(q) and (ba)-(bu).Any convenient signaling chromophore can be attached to any convenientpolymer dye described herein via coupling of compatible chemoselectivefunctional groups. The signaling chromophore can be selected to providefor a desirable emission spectra and emission maximum wavelength.

The subject polymeric dyes can include a sidechain chemoselectivefunctional group to which any convenient moiety of interest can beconjugated. Co-monomers which include such a sidechain group can bereferred to herein as linking co-monomers. The polymeric dye can beconjugated to a signaling chromophore to produce a polymeric tandem dyehaving a desirable spectroscopic properties. In some instances, themultichromophore (e.g., of formulae (I), (II) or (VI)), includes athiophene-containing co-monomer (e.g., M² or M³) having a linkedchemoselective functional group or a linked acceptor chromophore. Insome instances, the multichromophore (e.g., of formulae (I), (II) and(V)), includes a co-monomer having the structure of one of formulae(XVIII)-(XIX):

wherein: W¹ is an alkyl, a substituted alkyl or a WSG (e.g., asdescribed herein); and Dye is a signaling chromophore. In certainembodiments, the co-monomer is linked to a signaling chromophore and hasone of the following structures:

As used herein, the terms “chemoselective functional group” and“chemoselective tag” are used interchangeably and refer to a functionalgroup that can selectively react with another compatible functionalgroup to form a covalent bond, in some cases, after optional activationof one of the functional groups. Any convenient chemoselective tag andconjugation chemistries can be adapted for use in the subjectmultichromophores. Chemoselective functional groups of interest include,but are not limited to, thiols and thiol-reactive groups such asmaleimide, iodoacetamide or vinyl sulfone, amines and carboxylic acidsor active esters thereof, as well as groups that can react with oneanother via Click chemistry, e.g., azide and alkyne groups (e.g.,cyclooctyne groups), sulfur(VI) fluoride exchange chemistry (SuFEX),sulfonyl fluoride, tetrazine, alkyne, as well as aldehyde, hydroxyl,hydrazido, hydrazino, aldehyde, ketone, alkoxylamine, phosphine,epoxide, and the like.

Any convenient linking co-monomers may be incorporated into the subjectmultichromophores to provide for a linking group to which may beattached any convenient moieties of interest (e.g., a linked signalingchromophore). Linking co-monomers of interest include, but are notlimited to, those co-monomers described in the formula herein (e.g.,formula (XVIII)-(XIX)), a fluorene co-monomer, a phenylenevinyleneco-monomer, a phenyleneethynylene co-monomer, a carbazole co-monomer, aC₂-C₁₂ alkyne co-monomer, an arylene-ethynylene co-monomer, aheteroarylene-ethynylene co-monomer, an arylene co-monomer and aheteroarylene co-monomer. As used herein, the terms aryl or heteroarylco-monomer and arylene or heteroarylene co-monomer are usedinterchangeably. In certain cases, the linking co-monomer is athiophene-containing co-monomer (e.g., as described herein). In certaincases, the linking co-monomer is a substituted aryl or heteroarylco-monomer, such as a fluorene co-monomer (e.g., as described herein).

In some instances of any of the formula described herein, the signalingchromophore is linked to a co-monomer comprising 1% to 50% by molarityof the multichromophore, such as 1% to 20%, 1% to 10%, or 11 to 20% bymolarity. In certain cases, the multichromophore is a conjugated polymercomprising 5 or more repeat units.

Any convenient chemoselective functional groups may be included in thesubject multichromophores (e.g., at the —Z¹, —Z² and/or in the G¹ or G²terminal groups, including, but are not limited to, carboxylic acid,active ester (e.g., NHS or sulfo-NHS ester), amino, hydroxyl, thiol,maleimide, iodoacetyl, hydrazido, hydrazino, aldehyde, ketone, azido,alkyne, phosphine and epoxide. In some cases, the chemoselectivefunctional group or linker can be connected directed to a terminalco-monomer. It is understood that in the polymeric tandem dye structuresdescribed herein, in some cases, the groups Z¹ and Z² appear at aequivalent position in the structure where these groups can be usedinterchangeably to refer to either a linked signaling chromophore or achemoselective functional group that is capable of subsequentconjugation to a convenient chromophore precursor to produce the linkedsignaling chromophore.

In some cases, the signaling chromophore is a fluorophore. In certaincases, the signaling chromophore is a quencher. Any convenientfluorescent dyes may be utilized in the polymeric tandem dyes as anacceptor chromophore. The terms “fluorescent dye” and “fluorophore” areused interchangeably herein. The signaling chromophore (Z¹) can be a dyemolecule selected from a rhodamine, a coumarin, a xanthene, a cyanine, apolymethine, a pyrene, a dipyrromethene borondifluoride, a napthalimide,a phycobiliprotein, a peridinum chlorophyll protein, conjugates thereof,and combinations thereof. In certain embodiments, the signalingchromophore (Z¹) is a cyanine dye, a xanthene dye, a coumarin dye, athiazine dye and an acridine dye. In some instances, the signalingchromophore (Z¹) is selected from DY 431, DY 485XL, DY 500XL, DY 610, DY640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY734, DY 752, DY 778, DY 782, DY 800, DY 831, Biotium CF 555, Cy 3.5 anddiethylamino coumarin. In some embodiments, the acceptor chromophore isa cyanine dye, a xanthene dye, a coumarin dye, a thiazine dye or anacridine dye. Fluorescent dyes of interest include, but are not limitedto, fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine,carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine110, Cascade Blue, Cascade Yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5,Cy5.5, Cy-Chrome, phycoerythrin, PerCP (peridinin chlorophyll-aProtein), PerCP-Cy5.5, JOE(6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX(5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue,Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350,Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546,Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647,Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700,7-amino-4-methylcoumarin-3-acetic acid, BODIPY FL, BODIPY FL-Br.sub.2,BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY581/591, BODIPY 630/650, BODIPY 650/665, BODIPY R6G, BODIPY TMR, BODIPYTR, conjugates thereof, and combinations thereof. Lanthanide chelates ofinterest include, but are not limited to, europium chelates, terbiumchelates and samarium chelates. In some embodiments, the polymerictandem dye includes a polymeric dye linked to an acceptor fluorophoreselected from Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Alexa488, Alexa 647 andAlexa700. In certain embodiments, the polymeric tandem dye includes apolymeric dye linked to an acceptor fluorophore selected from Dyomicsdyes (such as DY 431, DY 485XL, DY 500XL, DY 530, DY 610, DY 633, DY640, DY 651, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY732, DY 734, DY 752, DY 754, DY 778, DY 782, DY 800 or DY 831), BiotiumCF 555, Cy 3.5, and diethylamino coumarin.

In some embodiments, the signaling chromophore that is selected has anemission maximum wavelength in the range of 300 to 900 nm, such as 350to 850 nm, 350 to 600 nm, 360 to 500 nm, 370 to 500 nm, 380 to 500 nm,390 to 500 nm or 400 to 500 nm, where specific examples of emissionmaxima of signaling chromophore of interest include, but are not limitedto: 395 nm±5 nm, 420 nm±5 nm, 430 nm±5 nm, 440 nm±5 nm, 450 nm±5 nm, 460nm±5 nm, 470 nm±5 nm, 480 nm±5 nm, 490 nm±5 nm, 500 nm±5 nm, 510 nm±5nm, 520 nm±5 nm, 530 nm±5 nm, 540 nm±5 nm, 550 nm±5 nm, 560 nm±5 nm, 570nm±5 nm, 580 nm±5 nm, 590 nm±5 nm, 605 nm±5 nm, 650 nm±5 nm, 680 nm±5nm, 700 nm±5 nm, 805 nm±5 nm.

End Groups

Any convenient end groups (e.g., G¹ and G²) may be utilized at theterminals of the subject multichromophores. As used herein, the terms“end group” and “terminal group” are used interchangeably to refer tothe groups located at the terminals of the polymeric structure of themultichromophore, e.g., as described herein. G¹ and G² groups ofinterest include, but are not limited to a terminal capping group, a πconjugated segment, a linker and a linked specific binding member. Insome embodiments, a terminal capping groups is a monovalent group whichis conjugated to the backbone of the multichromophore afterpolymerization. In certain instances, the terminal capping group is anaryl, a substituted aryl, a heteroaryl, a substituted heteroaryl, analkyl or a substituted alkyl. In certain cases, the terminal cappinggroup is derived from a monomer used in the method of polymerization,e.g., a terminal group such as a halogen (e.g., Br), a boronic acid or aboronic ester, which is capable of undergoing further conjugation. Insome instances, G¹ and/or G² is a π conjugated segment. As used herein,a π conjugated segment refers to any convenient segment of a conjugatedpolymer to which the multichromophore may be conjugated, i.e., allowingdelocalization of pi electron across adjacent units. In certainembodiments, G¹ and/or G² is a linker, such as a linker including afunctional group suitable for conjugation to a specific binding moiety.It is understood that linkers located at the G¹ and/or G² positions ofthe multichromophore may be selected so as to be orthogonal to any otherlinkers including chemoselective tags that may be present at a sidechainof the multichromophore (e.g., at Z²). In certain embodiments, an aminofunctional group or derivative thereof is included at G¹ and/or G² and acarboxylic acid functional group or derivative thereof is included atZ². In certain embodiments, a carboxylic acid functional group orderivative thereof is included at G¹ and/or G² and an amino functionalgroup or derivative thereof is included at Z².

In some embodiments of the formulae described herein, at least one of G¹and G² is -L³-Z⁴ where L³ is a linker (e.g., as described herein) and Z⁴is a specific binding member (e.g., as described herein). In someembodiments of formulae described herein, at least one of G¹ and G² is-L³-Z³ where L³ is a linker (e.g., as described herein) and Z³ is achemoselective tag (e.g., as described herein). In some instances, Z³ isselected from carboxylic acid, active ester (e.g., N-hydroxysuccinimidyl ester (NHS) or sulfo-NHS), amino, maleimide, iodoacetyl andthiol. In some instances, Z³ is a chemoselective functional group thatfinds us in a bioorthogonal conjugation chemistry, such as an azide oran alkyne (e.g., a cyclooctyne), sulfur(VI) fluoride exchange chemistry(SuFEX), sulfonyl fluoride or a tetrazine. In certain embodiments offormulae described herein, at least one of G¹ and G² is described by thefollowing structure:

*—Ar-L-Z

where Ar is a π-conjugated aryl group, L is a linker and Z is achemoselective tag or a specific binding member. In some cases, the L-Zgroup can be connected directed to a terminal co-monomer. In certainembodiments of formulae described herein, at least one of G¹ and G² isdescribed by the following structure:

wherein:

q is 0 or an integer from 1-12;

L is an optional linker; and

Z is a chemoselective tag or a specific binding member.

In certain embodiments, Z is a biomolecule. Biomolecules of interestinclude, but are not limited to, polypeptides, polynucleotides,carbohydrates, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs thereof and combinations thereof. In certaininstances, Z is an antibody. In some instances, Z is an antibodyfragment or binding derivative thereof. In some cases, the antibodyfragment or binding derivative thereof is selected from the groupconsisting of a Fab fragment, a F(ab′)₂ fragment, a scFv, a diabody anda triabody. Z can be any convenient chemoselective tag. In someinstances, Z is a chemoselective functional group that finds use in abioorthogonal Click chemistry, such as an azide or an alkyne (e.g., acyclooctyne), a tetrazine or an alkyne, or a sulfonyl fluoride. In somecases, Z is selected from carboxylic acid, active ester (e.g., N-hydroxysuccinimidyl ester (NHS) or sulfo-NHS), amino, hydroxyl, hydrazido,hydrazino, aldehyde, ketone, alkoxylamine, azido, alkyne, phosphine,epoxide maleimide, iodoacetyl and thiol.

It is understood that for any of the structures and formula depictedherein that in some cases of the subject multichromophore the end groupsdepicted may be located at the opposite ends to those shown, e.g., theend groups G¹ and -Ph-L-Z may be switched. In some embodiments of themultichromophores described herein (e.g., formulae (I)-(VIb), at leastone of G¹ and G² is selected from one of the following structures 1-33:

*=site for covalent attachment to unsaturated backbone;wherein R′ is independently H, halogen, C₁-C₁₂alkyl, (C₁-C₁₂alkyl)NH₂,C₂-C₁₂alkene, C₂-C₁₂alkyne, C₃-C₁₂cycloalkyl, C₁-C₁₂haloalkyl,C₂-C₁₈(hetero)aryl, C₂-C₁₈(hetro)arylamino, —[CH₂-CH₂]_(r)—Z¹, or(C₁-C₁₂)alkoxy-X¹; and wherein Z¹ is —OH or —COOH; X¹ is —NH₂, —NHCOOH,—NHCOOC(CH₃)₃, —NHCO(C3-C12)cycloalkyl(C1-C4)alkyl-N-maleimide; or—NHCO[CH₂-CH₂—O]_(s), (CH₂)_(s),NH₂; r′ is an integer from 1 to 20; andeach s′ is independently an integer from 1 to 20,(CH₂)₃(OCH₂CH₂)_(x″)OCH₃ where x″ is independently an integer from 0 to50, or a benzyl optionally substituted with one or more halogen,hydroxyl, C₁-C₁₂alkoxy, or (OCH₂CH₂)_(y″)CH₃where each y″ isindependently an integer from 0 to 50 and R′ is different from R;wherein k is 2, 4, 8, 12 or 24;wherein R¹⁵ is selected from the group consisting of I-u having thestructure:

*=site for covalent attachment to backbone.

In some embodiments of the multichromophores described herein (e.g.,formulae formulae (I)-(VIb), at least one end group (e.g., T²-Z², G¹,G², -L-Z, -L³-Z), or sidechain group, is selected from one of thefollowing structures:

wherein r is 0 or an integer from 1-50; k is 0 or an integer from 1-50(e.g., 1-20); R¹ is as defined for any of the fluorene co-monomersdescribed herein; and R¹⁶ is selected from H, OH, NH₂, —NH(CH₂)r—NH₂,and —NH(CH₂)_(r)COOH. In certain instances, r is 1 to 20, such as 3 to20, 3 to 15, 3 to 12, or 6 to 12. In certain cases, r is 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11 or 12. In some cases, r is 3. In some cases, r is 4.In some cases, r is 5. In some cases, r is 6. In some cases, r is 7. Insome cases, r is 8. In some cases, r is 9. In some cases, r is 10. Insome cases, r is 11. In some embodiments of the multichromophoresdescribed herein (e.g., formulae (I)-(VIb), at least one end group(e.g., T²-Z², G¹, G², -L-Z, -L³-Z) is

Labelled Specific Binding Members

Aspects of the present disclosure include labelled specific bindingmembers. A labelled specific binding member is a conjugate of a subjectpolymeric dye (e.g., as described herein) and a specific binding member.Any of the polymeric dyes and polymeric tandem dyes described herein maybe conjugated to a specific binding member. The specific binding memberand the polymeric dye can be conjugated (e.g., covalently linked) toeach other at any convenient locations of the two molecules, via anoptional linker. In certain instances, at least one of the terminalgroups of the polymeric dye (e.g., G¹ or G²) includes a linked specificbinding member.

As used herein, the term “specific binding member” refers to one memberof a pair of molecules which have binding specificity for one another.One member of the pair of molecules may have an area on its surface, ora cavity, which specifically binds to an area on the surface of, or acavity in, the other member of the pair of molecules. Thus the membersof the pair have the property of binding specifically to each other toproduce a binding complex. In some embodiments, the affinity betweenspecific binding members in a binding complex is characterized by aK_(d) (dissociation constant) of 10⁻⁶ M or less, such as 10⁻⁷ M or less,including 10⁻⁸ M or less, e.g., 10⁻⁹ M or less, 10⁻¹⁰ M or less, 10⁻¹¹ Mor less, 10⁻¹² M or less, 10⁻¹³ M or less, 10⁻¹⁴ M or less, including10⁻¹⁵ M or less. In some embodiments, the specific binding membersspecifically bind with high avidity. By high avidity is meant that thebinding member specifically binds with an apparent affinitycharacterized by an apparent K_(d) of 10×10⁻⁹ M or less, such as 1×10⁻⁹M or less, 3×10⁻¹⁰ M or less, 1×10⁻¹⁰ M or less, 3×10⁻¹¹ M or less,1×10⁻¹¹ M or less, 3×10⁻¹² M or less or 1×10⁻¹² M or less.

The specific binding member can be proteinaceous. As used herein, theterm “proteinaceous” refers to a moiety that is composed of amino acidresidues. A proteinaceous moiety can be a polypeptide. In certain cases,the proteinaceous specific binding member is an antibody. In certainembodiments, the proteinaceous specific binding member is an antibodyfragment, e.g., a binding fragment of an antibody that specific binds toa polymeric dye. As used herein, the terms “antibody” and “antibodymolecule” are used interchangeably and refer to a protein consisting ofone or more polypeptides substantially encoded by all or part of therecognized immunoglobulin genes. The recognized immunoglobulin genes,for example in humans, include the kappa (k), lambda (I), and heavychain genetic loci, which together comprise the myriad variable regiongenes, and the constant region genes mu (u), delta (d), gamma (g), sigma(e), and alpha (a) which encode the IgM, IgD, IgG, IgE, and IgA isotypesrespectively. An immunoglobulin light or heavy chain variable regionconsists of a “framework” region (FR) interrupted by three hypervariableregions, also called “complementarity determining regions” or “CDRs”.The extent of the framework region and CDRs have been precisely defined(see, “Sequences of Proteins of Immunological Interest,” E. Kabat etal., U.S. Department of Health and Human Services, (1991)). Thenumbering of all antibody amino acid sequences discussed herein conformsto the Kabat system. The sequences of the framework regions of differentlight or heavy chains are relatively conserved within a species. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDRs. The CDRs are primarily responsible for binding to an epitopeof an antigen. The term antibody is meant to include full lengthantibodies and may refer to a natural antibody from any organism, anengineered antibody, or an antibody generated recombinantly forexperimental, therapeutic, or other purposes as further defined below.

Antibody fragments of interest include, but are not limited to, Fab,Fab′, F(ab′)2, Fv, scFv, or other antigen-binding subsequences ofantibodies, either produced by the modification of whole antibodies orthose synthesized de novo using recombinant DNA technologies. Antibodiesmay be monoclonal or polyclonal and may have other specific activitieson cells (e.g., antagonists, agonists, neutralizing, inhibitory, orstimulatory antibodies). It is understood that the antibodies may haveadditional conservative amino acid substitutions which havesubstantially no effect on antigen binding or other antibody functions.

In certain embodiments, the specific binding member is a Fab fragment, aF(ab′)₂ fragment, a scFv, a diabody or a triabody. In certainembodiments, the specific binding member is an antibody. In some cases,the specific binding member is a murine antibody or binding fragmentthereof. In certain instances, the specific binding member is arecombinant antibody or binding fragment thereof.

In some embodiments, the labelled specific binding member includes: awater solvated light harvesting multichromophore (e.g., as describedherein); and a signaling chromophore covalently linked to themultichromophore in energy-receiving proximity therewith (e.g., asdescribed herein); and a specific binding member covalently linked tothe multichromophore. In some instances of the labelled specific bindingmember, the multichromophore of any of the formula described herein(e.g., formulae (V) and (VII)-(VIII)), wherein: G¹ and G² are eachindependently selected from a terminal group (e.g., end group), a πconjugated segment, a linker and a linked specific binding member,wherein at least one of G¹ and G² is a linked specific binding member.

Methods

As summarized above, aspects of the invention include methods ofevaluating a sample for the presence of a target analyte. Aspects of themethod include contacting the sample with a polymeric dye conjugate thatspecifically binds the target analyte to produce a labelling compositioncontacted sample. As used herein, the terms “polymeric dye conjugate”and “labelled specific binding member” are used interchangeably. Assuch, the polymeric dye conjugate can include: (i) a water solvatedpolymeric dye (e.g., as described herein); and (ii) a specific bindingmember (e.g., as described herein). In some instances, the polymeric dyeconjugate further comprises a signaling chromophore covalently linked toa multichromophore of the polymeric dye in energy-receiving proximitytherewith.

Any convenient method may be used to contact the sample with a polymericdye conjugate that specifically binds to the target analyte to producethe labelling composition contacted sample. In some instances, thesample is contacted with the polymeric dye conjugate under conditions inwhich the specific binding member specifically binds to the targetanalyte, if present. For specific binding of the specific binding memberof the conjugate with the target analyte, an appropriate solution may beused that maintains the biological activity of the components of thesample and the specific binding member. The solution may be a balancedsalt solution, e.g., normal saline, PBS, Hank's balanced salt solution,etc., conveniently supplemented with fetal calf serum, human plateletlysate or other factors, in conjunction with an acceptable buffer at lowconcentration, such as from 5-25 mM. Convenient buffers include HEPES,phosphate buffers, lactate buffers, etc. Various media are commerciallyavailable and may be used according to the nature of the target analyte,including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., in some casessupplemented with fetal calf serum or human platelet lysate. The finalcomponents of the solution may be selected depending on the componentsof the sample which are included.

The temperature at which specific binding of the specific binding memberof the conjugate to the target analyte takes place may vary, and in someinstances may range from 5° C. to 50° C., such as from 10° C. to 40° C.,15° C. to 40° C., 20° C. to 40° C., e.g., 20° C., 25° C., 30° C., 35° C.or 37° C. (e.g., as described above). In some instances, the temperatureat which specific binding takes place is selected to be compatible withthe biological activity of the specific binding member and/or the targetanalyte. In certain instances, the temperature is 25° C., 30° C., 35° C.or 37° C. In certain cases, the specific binding member is an antibodyor fragment thereof and the temperature at which specific binding takesplace is room temperature (e.g., 25° C.), 30° C., 35° C. or 37° C. Anyconvenient incubation time for specific binding may be selected to allowfor the formation of a desirable amount of binding complex, and in someinstances, may be 1 minute (min) or more, such as 2 min or more, 10 minor more, 30 min or more, 1 hour or more, 2 hours or more, or even 6hours or more.

Any convenient specific binding members may be utilized in theconjugate. Specific binding members of interest include, but are notlimited to, those agents that specifically bind cell surface proteins ofa variety of cell types, including but not limited to, stem cells, e.g.,pluripotent stem cells, hematopoietic stem cells, T cells, T regulatorcells, dendritic cells, B Cells, e.g., memory B cells, antigen specificB cells, granulocytes, leukemia cells, lymphoma cells, virus cells(e.g., HIV cells) NK cells, macrophages, monocytes, fibroblasts,epithelial cells, endothelial cells, and erythroid cells. Target cellsof interest include cells that have a convenient cell surface marker orantigen that may be captured by a convenient specific binding memberconjugate. In some embodiments, the target cell is selected from HIVcontaining cell, a Treg cell, an antigen-specific T -cell populations,tumor cells or hematopoetic progenitor cells (CD34+) from whole blood,bone marrow or cord blood. Any convenient cell surface proteins or cellmarkers may be targeted for specific binding to polymeric dye conjugatesin the subject methods. In some embodiments, the target cell includes acell surface marker selected from a cell receptor and a cell surfaceantigen. In some cases, the target cell may include a cell surfaceantigen such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25,CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34,CD1c, CD23, CD304, CD235a, T cell receptor alpha/beta, T cell receptorgamma/delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5(N-Terminal), SSEA-3, TRA-1-60 Antigen, Disialoganglioside GD2 and CD71.

Any convenient targets may be selected for evaluation utilizing thesubject methods. Targets of interest include, but are not limited to, anucleic acid, such as an RNA, DNA, PNA, CNA, HNA, LNA or ANA molecule, aprotein, such as a fusion protein, a modified protein, such as aphosphorylated, glycosylated, ubiquitinated, SUMOylated, or acetylatedprotein, or an antibody, a peptide, an aggregated biomolecule, a cell, asmall molecule, a vitamin and a drug molecule. As used herein, the term“a target protein” refers to all members of the target family, andfragments thereof. The target protein may be any protein of interest,such as a therapeutic or diagnostic target, including but not limitedto: hormones, growth factors, receptors, enzymes, cytokines,osteoinductive factors, colony stimulating factors and immunoglobulins.The term “target protein” is intended to include recombinant andsynthetic molecules, which can be prepared using any convenientrecombinant expression methods or using any convenient syntheticmethods, or purchased commercially. In some embodiments, the polymericdye conjugates include an antibody or antibody fragment. Any convenienttarget analyte that specifically binds an antibody or antibody fragmentof interest may be targeted in the subject methods.

In some embodiments, the target analyte is associated with a cell. Incertain instances, the target analyte is a cell surface marker of thecell. In certain cases, the cell surface marker is selected from thegroup consisting of a cell receptor and a cell surface antigen. In someinstances, the target analyte is an intracellular target, and the methodfurther includes lysing the cell.

In some embodiments, the sample may include a heterogeneous cellpopulation from which target cells are isolated. In some instances, thesample includes peripheral whole blood, peripheral whole blood in whicherythrocytes have been lysed prior to cell isolation, cord blood, bonemarrow, density gradient-purified peripheral blood mononuclear cells orhomogenized tissue. In some cases, the sample includes hematopoeticprogenitor cells (e.g., CD34+ cells) in whole blood, bone marrow or cordblood. In certain embodiments, the sample includes tumor cells inperipheral blood. In certain instances, the sample is a sample including(or suspected of including) viral cells (e.g., HIV).

The labelled specific binding members find use in the subject methods,e.g., for labeling a target cell, particle, target or analyte with apolymeric dye or polymeric tandem dye. For example, labelled specificbinding members find use in labeling cells to be processed (e.g.,detected, analyzed, and/or sorted) in a flow cytometer. The labelledspecific binding members may include antibodies that specifically bindto, e.g., cell surface proteins of a variety of cell types (e.g., asdescribed herein). The labelled specific binding members may be used toinvestigate a variety of biological (e.g., cellular) properties orprocesses such as cell cycle, cell proliferation, cell differentiation,DNA repair, T cell signaling, apoptosis, cell surface protein expressionand/or presentation, and so forth. Labelled specific binding members maybe used in any application that includes (or may include)antibody-mediated labeling of a cell, particle or analyte.

In some instances of the method, the labelled specific binding memberincludes a multichromophore according to any one of formulae (I)-(VI)(e.g., as described herein). In certain cases, G¹ and G² are eachindependently selected from the group consisting of a terminal group, aπ conjugated segment, a linker and a linked specific binding member,wherein at least one of G¹ and G² is a linked specific binding member.

Aspects of the method include assaying the labelling compositioncontacted sample for the presence of a polymeric dye conjugate-targetanalyte binding complex to evaluate whether the target analyte ispresent in the sample. Once the sample has been contacted with thepolymeric dye conjugate, any convenient methods may be utilized inassaying the labelling composition contacted sample that is produced forthe presence of a polymeric dye conjugate-target analyte bindingcomplex. The polymeric dye conjugate-target analyte binding complex isthe binding complex that is produced upon specific binding of thespecific binding member of the conjugate to the target analyte, ifpresent. Assaying the labelling composition contacted sample can includedetecting a fluorescent signal from the binding complex, if present. Insome cases, the assaying includes a separating step where the targetanalyte, if present, is separated from the sample. A variety of methodscan be utilized to separate a target analyte from a sample, e.g., viaimmobilization on a support. Assay methods of interest include, but arenot limited to, any convenient methods and assay formats where pairs ofspecific binding members such as avidin-biotin or hapten-anti-haptenantibodies find use, are of interest. Methods and assay formats ofinterest that may be adapted for use with the subject compositionsinclude, but are not limited to, flow cytometry methods, in-situhybridization methods, enzyme-linked immunosorbent assays (ELISAs),western blot analysis, magnetic cell separation assays and fluorochromepurification chromatography.

In certain embodiments, the method further includes contacting thesample with a second specific binding member that specifically binds thetarget analyte. In certain instances, the second specific binding memberis support bound. Any convenient supports may be utilized to immobilizea component of the subject methods (e.g., a second specific bindingmember). In certain instances, the support is a particle, such as amagnetic particle. In some instances, the second specific binding memberand the polymeric dye conjugate produce a sandwich complex that may beisolated and detected, if present, using any convenient methods. In someembodiments, the method further includes flow cytometrically analyzingthe polymeric dye conjugate-target analyte binding complex, i.e., afluorescently labelled target analyte. Assaying for the presence of apolymeric dye conjugate-target analyte binding complex may provide assayresults (e.g., qualitative or quantitative assay data) which can be usedto evaluate whether the target analyte is present in the sample.

Any convenient supports may be utilized in the subject methods toimmobilize any convenient component of the methods, e.g., labelledspecific binding member, target, secondary specific binding member, etc.Supports of interest include, but are not limited to: solid substrates,where the substrate can have a variety of configurations, e.g., a sheet,bead, or other structure, such as a plate with wells; beads, polymers,particle, a fibrous mesh, hydrogels, porous matrix, a pin, a microarraysurface, a chromatography support, and the like. In some instances, thesupport is selected from the group consisting of a particle, a planarsolid substrate, a fibrous mesh, a hydrogel, a porous matrix, a pin, amicroarray surface and a chromatography support. The support may beincorporated into a system that it provides for cell isolation assistedby any convenient methods, such as a manually-operated syringe, acentrifuge or an automated liquid handling system. In some cases, thesupport finds use in an automated liquid handling system for the highthroughput isolation of cells, such as a flow cytometer.

In some embodiments of the method, the separating step includes applyingan external magnetic field to immobilize a magnetic particle. Anyconvenient magnet may be used as a source of the external magnetic field(e.g., magnetic field gradient). In some cases, the external magneticfield is generated by a magnetic source, e.g. by a permanent magnet orelectromagnet. In some cases, immobilizing the magnetic particles meansthe magnetic particles accumulate near the surface closest to themagnetic field gradient source, i.e. the magnet.

The separating may further include one or more optional washing steps toremove unbound material of the sample from the support. Any convenientwashing methods may be used, e.g., washing the immobilized support witha biocompatible buffer which preserves the specific binding interactionof the polymeric dye and the specific binding member. Separation andoptional washing of unbound material of the sample from the supportprovides for an enriched population of target cells where undesiredcells and material may be removed.

In certain embodiments, the method further includes detecting thelabelled target. Detecting the labelled target may include exciting themultichromophore with one or more lasers and subsequently detectingfluorescence emission from the polymeric tandem dye using one or moreoptical detectors. Detection of the labelled target can be performedusing any convenient instruments and methods, including but not limitedto, flow cytometry, FACS systems, fluorescence microscopy; fluorescence,luminescence, ultraviolet, and/or visible light detection using a platereader; high performance liquid chromatography (HPLC); and massspectrometry. When using fluorescently labeled components in the methodsand compositions of the present disclosure, it is recognized thatdifferent types of fluorescence detection systems can be used topractice the subject methods. In some cases, high throughput screeningcan be performed, e.g., systems that use 96 well or greater microtiterplates. A variety of methods of performing assays on fluorescentmaterials can be utilized, such as those methods described in, e.g.,Lakowicz, J. R., Principles of Fluorescence Spectroscopy, New York:Plenum Press (1983); Herman, B., Resonance energy transfer microscopy,in: Fluorescence Microscopy of Living Cells in Culture, Part B, Methodsin Cell Biology, vol. 30, ed. Taylor, D. L. & Wang, Y. -L., San Diego:Academic Press (1989), pp. 219-243; Turro, N.J., Modern MolecularPhotochemistry, Menlo Park: Benjamin/Cummings Publishing Col, Inc.(1978), pp. 296-361.

Fluorescence in a sample can be measured using a fluorimeter. In somecases, excitation radiation, from an excitation source having a firstwavelength, passes through excitation optics. The excitation opticscause the excitation radiation to excite the sample. In response,fluorescently labelled targets in the sample emit radiation which has awavelength that is different from the excitation wavelength. Collectionoptics then collect the emission from the sample. The device can includea temperature controller to maintain the sample at a specifictemperature while it is being scanned. In certain instances, amulti-axis translation stage moves a microtiter plate holding aplurality of samples in order to position different wells to be exposed.The multi-axis translation stage, temperature controller, auto-focusingfeature, and electronics associated with imaging and data collection canbe managed by an appropriately programmed digital computer. The computeralso can transform the data collected during the assay into anotherformat for presentation.

In some embodiments, the method of evaluating a sample for the presenceof a target analyte further includes detecting fluorescence in a flowcytometer. In some embodiments, the method of evaluating a sample forthe presence of a target analyte further includes imaging the labellingcomposition contacted sample using fluorescence microscopy. Fluorescencemicroscopy imaging can be used to identify a polymeric dyeconjugate-target analyte binding complex in the contacted sample toevaluate whether the target analyte is present. Microscopy methods ofinterest that find use in the subject methods include laser scanningconfocal microscopy.

Also provided are methods of labelled a target molecule. The subjectpolymeric dyes, find use in a variety of methods of labelling,separation, detection and/or analysis. In some embodiments, the methodincludes: contacting the target molecule with a polymeric dye (e.g., asdescribed herein) to produce a labelled target molecule, wherein thepolymeric dye includes a conjugation tag that covalently links to thetarget molecule. In some cases, the polymeric dye further includes asignaling chromophore covalently linked to the multichromophore of thepolymeric dye in energy-receiving proximity therewith. In some instancesof the method, the polymeric dye member includes a multichromophoreaccording to any one of formulae (I)-(VI) (e.g., as described herein),where one of G¹ and G² is a terminal group and the other of G¹ and G² isthe conjugation tag.

As used herein the term “conjugation tag” refers to a group thatincludes a chemoselective functional group (e.g., as described herein)that can covalently link with a compatible functional group of a targetmolecule, after optional activation and/or deprotection. Any convenientconjugation tags may be utilized in the subject polymeric dyes in orderto conjugate the dye to a target molecule of interest. In someembodiments, the conjugation tag includes a terminal functional groupselected from an amino, a carboxylic acid or a derivative thereof, athiol, a hydroxyl, a hydrazine, a hydrazide, a azide, an alkyne and aprotein reactive group (e.g. amino-reactive, thiol-reactive,hydroxyl-reactive, imidazolyl-reactive or guanidinyl-reactive).

Any convenient methods and reagent may be adapted for use in the subjectlabelling methods in order to covalently link the conjugation tag to thetarget molecule. Methods of interest for labelling a target, include butare not limited to, those methods and reagents described by Hermanson,Bioconjugate Techniques, Third edition, Academic Press, 2013. Thecontacting step may be performed in an aqueous solution. In someinstances, the conjugation tag includes an amino functional group andthe target molecule includes an activated ester functional group, suchas a NHS ester or sulfo-NHS ester, or vice versa. In certain instances,the conjugation tag includes a maleimide functional group and the targetmolecule includes a thiol functional group, or vice versa. In certaininstances, the conjugation tag includes an alkyne (e.g., a cyclooctynegroup) functional group and the target molecule includes an azidefunctional group, or vice versa, which can be conjugated via Clickchemistry.

Any convenient target molecules may be selected for labelling utilizingthe subject methods. Target molecules of interest include, but are notlimited to, a nucleic acid, such as an RNA, DNA, PNA, CNA, HNA, LNA orANA molecule, a protein, such as a fusion protein, a modified protein,such as a phosphorylated, glycosylated, ubiquitinated, SUMOylated, oracetylated protein, or an antibody, a peptide, an aggregatedbiomolecule, a cell, a small molecule, a vitamin and a drug molecule. Asused herein, the term “a target protein” refers to all members of thetarget family, and fragments thereof. The target protein may be anyprotein of interest, such as a therapeutic or diagnostic target,including but not limited to: hormones, growth factors, receptors,enzymes, cytokines, osteoinductive factors, colony stimulating factorsand immunoglobulins. The term “target protein” is intended to includerecombinant and synthetic molecules, which can be prepared using anyconvenient recombinant expression methods or using any convenientsynthetic methods, or purchased commercially. In some embodiments, thetarget molecule is a specific binding member (e.g., as describedherein). In certain instances, the specific binding member is anantibody. In some instances, the specific binding member is an antibodyfragment or binding derivative thereof. In some case, the antibodyfragment or binding derivative thereof is selected from the groupconsisting of a Fab fragment, a F(ab′)₂ fragment, a scFv, a diabody anda triabody.

In some cases, the method includes a separating step where the labelledtarget molecule is separated from the reaction mixture, e.g., excessreagents or unlabeled target. A variety of methods may be utilized toseparate a target from a sample, e.g., via immobilization on a support,precipitation, chromatography, and the like.

In some instances, the method further includes detecting and/oranalyzing the labelled target molecule. In some instances, the methodfurther includes fluorescently detecting the labelled target molecule.Any convenient methods may be utilized to detect and/or analyze thelabelled target molecule in conjunction with the subject methods andcompositions. Methods of analyzing a target of interest that find use inthe subject methods, include but are not limited to, flow cytometry,fluorescence microscopy, in-situ hybridization, enzyme-linkedimmunosorbent assays (ELISAs), western blot analysis, magnetic cellseparation assays and fluorochrome purification chromatography.Detection methods of interest include but are not limited tofluorescence spectroscopy, fluorescence microscopy, nucleic acidsequencing, fluorescence in-situ hybridization (FISH), protein massspectroscopy, flow cytometry, and the like.

Detection may be achieved directly via the polymeric dye or polymerictandem dye, or indirectly by a secondary detection system. The lattermay be based on any one or a combination of several different principlesincluding, but not limited to, antibody labelled anti-species antibodyand other forms of immunological or non-immunological bridging andsignal amplification systems (e.g., biotin-streptavidin technology,protein-A and protein-G mediated technology, or nucleic acidprobe/anti-nucleic acid probes, and the like). Suitable reportermolecules may be those known in the field of immunocytochemistry,molecular biology, light, fluorescence, and electron microscopy, cellimmunophenotyping, cell sorting, flow cytometry, cell visualization,detection, enumeration, and/or signal output quantification. More thanone antibody of specific and/or non-specific nature might be labelledand used simultaneously or sequentially to enhance target detection,identification, and/or analysis.

Systems

Aspects of the invention further include systems for use in practicingthe subject methods and compositions. A sample analysis system caninclude sample field of view or a flow channel loaded with a sample anda labelled specific binding member. In some embodiments, the system is aflow cytometric system including: a flow cytometer including a flowpath; a composition in the flow path, wherein the composition includes:a sample; and a labelled specific binding member (e.g., as describedherein).

In some embodiments, the system for analyzing a sample is a fluorescencemicroscopy system, including: a fluorescence microscope comprising asample field of view; and a composition disposed in the sample field ofview, wherein the composition comprises a sample; and a labelledspecific binding member (e.g., as described herein).

In some instances of the systems, the labelled specific binding memberincludes: a water solvated light harvesting multichromophore (e.g., asdescribed herein) and a specific binding member that specifically bindsa target analyte covalently linked to the multichromophore. In somecases, the labelled specific binding member further comprises asignaling chromophore covalently linked to the multichromophore of thepolymeric dye in energy-receiving proximity therewith. In some instancesof the subject systems, the labelled specific binding member, themultichromophore is described by any one of formulae (I)-(VI) (e.g., asdescribed herein), wherein: G¹ and G² are each independently selectedfrom the group consisting of a terminal group, a π conjugated segment, alinker and a linked specific binding member, wherein at least one of G¹and G² is a linked specific binding member.

In certain embodiments of the systems, the composition further includesa second specific binding member that is support bound and specificallybinds the target analyte. In some cases, the support includes a magneticparticle. As such, in certain instances, the system may also include acontrollable external paramagnetic field configured for application toan assay region of the flow channel.

The sample may include a cell. In some instances, the sample is acell-containing biological sample. In some instances, the sampleincludes a labelled specific binding member specifically bound to atarget cell. In certain instances, the target analyte that isspecifically bound by the specific binding member is a cell surfacemarker of the cell. In certain cases, the cell surface marker isselected from the group consisting of a cell receptor and a cell surfaceantigen.

In certain aspects, the system may also include a light sourceconfigured to direct light to an assay region of the flow channel orsample field of view. The system may include a detector configured toreceive a signal from an assay region of the flow channel or a samplefield of view, wherein the signal is provided by the fluorescentcomposition. Optionally further, the sample analysis system may includeone or more additional detectors and/or light sources for the detectionof one or more additional signals.

In certain aspects, the system may further include computer-basedsystems configured to detect the presence of the fluorescent signal. A“computer-based system” refers to the hardware means, software means,and data storage means used to analyze the information of the presentinvention. The minimum hardware of the computer-based systems of thepresent invention includes a central processing unit (CPU), input means,output means, and data storage means. A skilled artisan can readilyappreciate that any one of the currently available computer-based systemare suitable for use in the subject systems. The data storage means mayinclude any manufacture including a recording of the present informationas described above, or a memory access means that can access such amanufacture.

To “record” data, programming or other information on a computerreadable medium refers to a process for storing information, using anysuch methods as known in the art. Any convenient data storage structuremay be chosen, based on the means used to access the stored information.A variety of data processor programs and formats can be used forstorage, e.g., word processing text file, database format, etc.

A “processor” references any hardware and/or software combination thatwill perform the functions required of it. For example, any processorherein may be a programmable digital microprocessor such as available inthe form of an electronic controller, mainframe, server or personalcomputer (desktop or portable). Where the processor is programmable,suitable programming can be communicated from a remote location to theprocessor, or previously saved in a computer program product (such as aportable or fixed computer readable storage medium, whether magnetic,optical or solid state device based). For example, a magnetic medium oroptical disk may carry the programming, and can be read by a suitablereader communicating with each processor at its corresponding station.

In addition to the sensor device and signal processing module, e.g., asdescribed above, systems of the invention may include a number ofadditional components, such as data output devices, e.g., monitorsand/or speakers, data input devices, e.g., interface ports, keyboards,etc., fluid handling components, power sources, etc.

In certain aspects, the system includes a flow cytometer. Flowcytometers of interest include, but are not limited, to those devicesdescribed in U.S. Pat. Nos. 4,704,891; 4,727,029; 4,745,285; 4,867,908;5,342,790; 5,620,842; 5,627,037; 5,701,012; 5,895,922; and 6,287,791;the disclosures of which are herein incorporated by reference.

Other systems may find use in practicing the subject methods. In certainaspects, the system may be a fluorimeter or microscope loaded with asample having a fluorescent composition of any of the embodimentsdiscussed herein. The fluorimeter or microscope may include a lightsource configured to direct light to the assay region of the flowchannel or sample field of view. The fluorimeter or microscope may alsoinclude a detector configured to receive a signal from an assay regionof the flow channel or field of view, wherein the signal is provided bythe fluorescent composition.

Kits

Aspects of the invention further include kits for use in practicing thesubject methods and compositions. The compositions of the invention canbe included as reagents in kits either as starting materials or providedfor use in, for example, the methodologies described above.

A kit can include a polymeric dye including a water solvated lightharvesting multichromophore (e.g., as described herein) and a container.Any convenient containers can be utilized, such as tubes, bottles, orwells in a multi-well strip or plate, a box, a bag, an insulatedcontainer, and the like. The subject kits can further include one ormore components selected from a polymeric tandem dye, a fluorophore, aspecific binding member, a specific binding member conjugate, a supportbound specific binding member, a cell, a support, a biocompatibleaqueous elution buffer, and instructions for use. In some embodiments ofthe kit, the multichromophore is covalently linked to a specific bindingmember. In some instances, the specific binding member is an antibody.In certain instances, the specific binding member is an antibodyfragment or binding derivative thereof. In certain cases, the antibodyfragment or binding derivative thereof is selected from the groupconsisting of a Fab fragment, a F(ab′)2 fragment, a scFv, a diabody anda triabody.

In certain embodiments, the kit finds use in evaluating a sample for thepresence of a target analyte, such as an intracellular target. As such,in some instances, the kit includes one or more components suitable forlysing cells. The one or more additional components of the kit may beprovided in separate containers (e.g., separate tubes, bottles, or wellsin a multi-well strip or plate).

In certain aspects, the kit further includes reagents for performing aflow cytometric assay. Reagents of interest include, but are not limitedto, buffers for reconstitution and dilution, buffers for contacting acell sample the multichromophore, wash buffers, control cells, controlbeads, fluorescent beads for flow cytometer calibration and combinationsthereof. The kit may also include one or more cell fixing reagents suchas paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or anycombinations or buffers thereof. Further, the kit may include a cellpermeabilizing reagent, such as methanol, acetone or a detergent (e.g.,triton, NP-40, saponin, tween 20, digitonin, leucoperm, or anycombinations or buffers thereof. Other protein transport inhibitors,cell fixing reagents and cell permeabilizing reagents familiar to theskilled artisan are within the scope of the subject kits.

The compositions of the kit may be provided in a liquid composition,such as any suitable buffer. Alternatively, the compositions of the kitmay be provided in a dry composition (e.g., may be lyophilized), and thekit may optionally include one or more buffers for reconstituting thedry composition. In certain aspects, the kit may include aliquots of thecompositions provided in separate containers (e.g., separate tubes,bottles, or wells in a multi-well strip or plate).

In addition, one or more components may be combined into a singlecontainer, e.g., a glass or plastic vial, tube or bottle. In certaininstances, the kit may further include a container (e.g., such as a box,a bag, an insulated container, a bottle, tube, etc.) in which all of thecomponents (and their separate containers) are present. The kit mayfurther include packaging that is separate from or attached to the kitcontainer and upon which is printed information about the kit, thecomponents of the and/or instructions for use of the kit.

In addition to the above components, the subject kits may furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, DVD, portable flash drive, etc., on which theinformation has been recorded. Yet another means that may be present isa website address which may be used via the Internet to access theinformation at a removed site. Any convenient means may be present inthe kits.

Utility

The polymeric dyes, compositions, methods and systems as describedherein may find use in a variety of applications, including diagnosticand research applications, in which the labelling, detection and/oranalysis of a target of interest is desirable. The subject polymericdyes can be used as fluorescent labels in a variety of applications tobe utilized with excitation sources from 430 nm to 530 nm. A key lasersource used in a variety of biodetection techniques is the Argon (Ar)ion laser used as a continuous wave source of 488 nm light. Because ofthe early availability of such laser sources, virtually all flowcytometers have a 488 nm source. Thus subject polymeric dyes that exciteat 488 nm are of interest and can find use in flow cytometryinstrumentation and experimental setups. Additionally, Ar ion lasershave several lines close to 488 nm with significant intensity. Coupledwith the availability of a wide range of wavelengths for modern diodelasers and the central location of this range within the UV-visiblespectrum, the region from 440 nm to 530 nm is of interest for excitationof the subject polymeric dyes.

Applications of interest include, but are not limited to, methodologiessuch as cytometry, microscopy, immunoassays (e.g. competitive ornon-competitive), assessment of a free analyte, assessment of receptorbound ligand, and so forth. The compositions, system and methodsdescribed herein may be useful in analysis of any of a number ofsamples, including but not limited to, biological fluids, cell culturesamples, and tissue samples. In certain aspects, the compositions,system and methods described herein may find use in methods whereanalytes are detected in a sample, if present, using fluorescent labels,such as in fluorescent activated cell sorting or analysis, immunoassays,immuno-staining, and the like. In certain instances, the compositionsand methods find use in applications where the evaluation of a samplefor the presence of a target analyte is of interest.

In some cases, the methods and compositions find use in any assay formatwhere the detection and/or analysis of a target from a sample is ofinterest, including but not limited to, flow cytometry, fluorescencemicroscopy, in-situ hybridization, enzyme-linked immunosorbent assays(ELISAs), western blot analysis, magnetic cell separation assays andfluorochrome purification chromatography. In certain instances, themethods and compositions find use in any application where thefluorescent labelling of a target molecule is of interest. The subjectcompositions may be adapted for use in any convenient applications wherepairs of specific binding members find use, such as biotin-streptavidinand hapten-anti-hapten antibody.

EXAMPLES Example 1 Synthesis of Conjugated Polymer via C—H BondArylation

The synthesis of the subject dyes can be achieved by a technique calledC—H bond arylation (Scheme 1). Other methods such as a Suzuki couplingmethod (Scheme 2) or a Stille coupling method (Scheme 3) can also beutilized. Unlike Suzuki coupling with involves synthesis of boronicester/acid monomers, or Stille coupling which involves toxic tinreagents, this method uses simpler monomers and achieves high molecularweights relative to other coupling reactions that can be employed. Theabsorption profiles of several exemplary dyes (see FIG. 1) that wereprepared according to the methods described herein are shown in FIG. 2.

Polymer Synthesis Procedure:

To a single-neck round bottom flask (with a side arm) charged with aTeflon coated stir bar, was added monomer A (446.2 mg, 0.1775 mmol),monomer B (212.9 mg, 0.1775 mmol), potassium carbonate (61.3 mg, 0.444mmol), Pd(OAc)₂ (2.0 mg), pivalic acid (5.4 mg), and dimethylacetamidesolvent (1.25 mL). The flask was equipped with a condenser and connectedto a Schenk line. The solution was degassed by nitrogen flushing andthree freeze-pump-thaw cycles. The mixture was then stirred at 80° C.overnight under to nitrogen protection.

The resulting polymer solution was cooled to room temperature, dilutedwith 20% EtOH/H₂O, and stirred with EDTA (60 mg) at 23° C. for 1 h. Themixture was spin filtered and rinsed with 20% EtOH/H₂O. Polymer wascollected after vacuum drying (550 mg). Mn=105 kDa; Mw=191 kDa;Absorption λ_(max)=471 nm; Fluorescence λ_(max)=516 nm.

Notwithstanding the appended claims, the disclosure set forth herein isalso defined by the following clauses:

-   Clause 1. A water solvated polymeric dye having a blue excitation    spectrum.-   Clause 2. The polymeric dye according to clause 1, wherein the    polymeric dye comprises a thiophene containing co-monomer.-   Clause 3. The polymeric dye according to any one of clauses 1-2,    wherein the polymeric dye has no yellow-green absorption at 562 nm.-   Clause 4. The polymeric dye according to any one of clauses 1-3,    wherein the excitation spectrum of the polymeric dye has a full    width at half maximum (FWHM) that is 100 nm or less in width.-   Clause 5. The polymeric dye according to any one of clauses 1-4,    wherein the polymeric dye has an absorption maximum wavelength of    488 nm±20 nm.-   Clause 6. The polymeric dye according to any one of clauses 1-5,    wherein the polymeric dye has an extinction coefficient of 1×10⁶ M⁻¹    cm⁻¹ or more.-   Clause 7. The polymeric dye according to any one of clauses 1-6,    wherein the polymeric dye is substituted with non-ionic side groups    capable of imparting solubility in water in excess of 10 mg/mL.-   Clause 8. The polymeric dye according to any one of clauses 1-7,    wherein the polymeric dye comprises a conjugated segment having the    structure of formula (I):

wherein:

each M¹, M² and M³ is independently an aryl or heterocyclic co-monomerwherein at least one of M¹, M² and M³ is a thiophene containingco-monomer;

Z¹ is a chemoselective functional group or a linked acceptorchromophore;

Z⁵ and Z⁶ are independently CR or N where R is H, halogen, alkoxy,substituted alkoxy, alkyl and substituted alkyl;

G¹ and G² are each independently selected from a terminal group, a πconjugated segment, a linker and a linked specific binding member;

Y¹, Y² and Y³ are independently C(R³)₂, —C(R³)₂C(R³)₂—, NR³, Si(R³)₂ orSe;

each R³ is independently selected from H, alkyl, substituted alkyl,aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl,substituted acyl, alkoxy, substituted alkoxy, amido, substituted amidoand a WSG (e.g., water soluble group);

a-i are independently 0 or 1;

each n, m and o are independently 0 or an integer from 1 to 10,000; and

p is an integer from 1 to 100,000.

-   Clause 9. The polymeric dye according to clause 8, wherein M¹, M²    and M³ are each independently a thiophene containing co-monomer.-   Clause 10. The polymeric dye according to any one of clauses 8-9,    wherein:

a+c≦1;

d+f≦1;

g+i ≦1;

b+e+h≧1; and

n+m+o≧1.

-   Clause 11. The polymeric dye according to any one of clauses 8-10,    wherein the polymeric dye has the structure of one of formula    (Ib)-(Ic):

-   Clause 12. The polymeric dye according to any one of clauses 8-11,    wherein the polymeric dye has the structure of one of formula    (IIa)-(IIb):

wherein u, v, w, x, y and z represent mol % values for each co-monomerin the multichromophore.

-   Clause 13. The polymeric dye according to any one of clauses 8-12,    wherein the polymeric dye has the structure of one of formula    (IIIa)-(IIIb):

wherein n is an integer from 1 to 100,000.

-   Clause 14. The polymeric dye according to clause 13, wherein Y¹ is    C(R³)₂.-   Clause 15. The polymeric dye according to clause 13, wherein Y¹ is    NR³.-   Clause 16. The polymeric dye according to any one of clauses 8-12,    wherein the polymeric dye has the structure of one of formula    (IVa)-(IVb):

wherein:

M¹ and M² are each independently a thiophene containing co-monomer;

each n and m are independently an integer from 1 to 10,000;

p is an integer from 1 to 100,000.

-   Clause 17. The polymeric dye according to clause 16, wherein: either

Y¹ is NR³; Y² is C(R³)₂; and M¹ and M² are the same thiophene containingco-monomer; or

Y¹ is C(R³)₂; Y² is NR³; and M¹ and M² are the same thiophene containingco-monomer.

-   Clause 18. The polymeric dye according to clause 16, wherein: Y¹ and    Y² are each independently C(R³)₂; and M¹ and M² are the same    thiophene containing co-monomer.-   Clause 19. The polymeric dye according to clause 8 or 11, wherein    the polymeric dye has the structure of one of formula (Va)-(Vb):

wherein:

M¹ and M³ are each independently a thiophene containing co-monomer;

each n and each o are independently an integer from 1 to 10,000; and

p is an integer from 1 to 100,000.

-   Clause 20. The polymeric dye according to clause 19, wherein Y³—Z₁    is C(R³)(T¹-Z¹), wherein Z¹ is a linked signaling chromophore and T¹    is a linker.-   Clause 21. The polymeric dye according to clause 19, wherein Y³—Z¹    is N-T¹-Z¹, wherein Z¹ is a linked signaling chromophore and T¹ is a    linker.-   Clause 22. The polymeric dye according to any one of clauses 19-21,    wherein: Y¹ is NR³; and M¹ and M³ are the same thiophene containing    co-monomer.-   Clause 23. The polymeric dye according to any one of clauses 19-21,    wherein: Y¹ is C(R³)₂; and M¹ and M³ are the same thiophene    containing co-monomer.-   Clause 24. The polymeric dye according to clause 8 or 11, wherein    the polymeric dye has the structure of one of formula (VIa)-(VIb):

wherein:

M¹ and M² are each independently a thiophene containing co-monomer;

each n and each m are independently an integer from 1 to 10,000;

Z¹ is a linked chemoselective functional group or a linked signalingchromophore;

and

-   -   p is an integer from 1 to 100,000.

-   Clause 25. The polymeric dye according to clause 24, wherein Y¹ and    Y² are NR³.

-   Clause 26. The polymeric dye according to clause 24, wherein Y¹ and    Y² are C(R³)₂.

-   Clause 27. The polymeric dye according to clause 24, wherein either    Y¹ is NR³ and Y² is C(R³)₂, or Y¹ is C(R³)₂ and Y² is NR³.

-   Clause 28. The polymeric dye according to any one of clauses 2-27,    wherein the thiophene-containing co-monomer is described by formula    (VII), (VIII) or (IX):

wherein:

A1 and A2 are each a fused monocyclic or bicyclic group

each R³ is independently selected from H, amino, substituted amino,halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido,substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy, WSGand -T²-Z², wherein Z² is a chemoselective functional group or a linkedsignaling chromophore, and T² is a linker;

n is 0, 1, 2, 3 or 4; and

each * is a site for covalent attachment to the unsaturated backbone ofa conjugated polymer.

-   Clause 29. The polymeric dye according to clause 28, wherein Al and    A2 are selected from a fused monocyclic heterocycle, a fused    monocyclic heteroaryl and a fused bicyclic heteroaryl.-   Clause 30. The polymeric dye according to any one of clauses 28-29,    wherein the thiophene-containing co-monomer is described by one of    formula (X)-(XVII):

wherein:

X¹ is CR³, NR³, O or S;

X² is N, O or S, wherein when X² is N, each

is a double bond and when X² is O or S, each

is a single bond;

R¹ and R² together form a 5- or 6-membered fused aryl or heteroaryl ringwhich is optionally substituted with one or more R³ groups; and

Y⁴ is NR³, C(R³)₂ or Si(R³)₂.

-   Clause 31. The polymeric dye according to clause 30, wherein the    thiophene-containing co-monomer is described by one of the following    structures (a) to (q):

wherein:

each R⁴ is independently selected from H, amino, substituted amino,halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido,substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy and-T²-Z², wherein Z² is a chemoselective functional group or a linkedacceptor chromophore, and T² is a linker; and

each s is 0 or an integer from 1 to 50.

-   Clause 32. The polymeric dye according to any one of clauses 8-31,    wherein each R³ and each R⁴ is independently selected from H,    substituted aryl, alkyl and the following structures:

wherein: each T⁵ is independently an optional linker; and each s is 0 oran integer from 1 to 50.

-   Clause 33. The polymeric dye according to any one of clauses 28-32,    wherein the thiophene-containing co-monomer is described by one of    the following structures (ba) to (bu):

-   Clause 34. The polymeric dye according to any one of clauses 8-33,    wherein the polymeric dye comprises a co-monomer linked to a    signaling chromophore having the structure of formula (XVI):

wherein: W is an alkyl, a substituted alkyl or a WSG; and Dye is asignaling chromophore.

-   Clause 35. The polymeric dye according to clause 34, wherein the    polymeric dye comprises a co-monomer linked to a signaling    chromophore having one of the following structures:

-   Clause 36. The polymeric dye according to any one of clauses 8-30,    wherein co-monomers M¹-M³ are independently selected from    formulae (XVIII) and (XIX):

wherein:

R¹ and R² together form a 5- or 6-membered fused aryl or heteroaryl ringwhich is optionally substituted with one or more R³ groups; and

each R³ is independently selected from H, amino, substituted amino,halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido,substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy and-T²-Z², wherein Z² is a chemoselective functional group or a linkedacceptor chromophore, and T² is a linker.

-   Clause 37. The polymeric dye according to any one of clauses 8-30,    wherein co-monomers M¹-M³ are independently selected from formulae    (e)-(i):

wherein:

each R⁴ is independently selected from H, amino, substituted amino,halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido,substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy and-T²-Z², wherein Z² is a chemoselective functional group or a linkedacceptor chromophore, and T² is a linker; and

each s is 0 or an integer from 1 to 50.

-   Clause 38. The polymeric dye according to any one of clauses 36-37,    wherein each R³ and each R⁴ is independently selected from H,    substituted aryl, alkyl and the following structures:

wherein:

each T⁵ is independently an optional linker; and

each s is 0 or an integer from 1 to 50.

-   Clause 39. A polymeric tandem dye comprising:

a polymeric dye having a blue excitation spectrum according to any oneof clauses 1-38; and

a signaling chromophore covalently linked to the polymeric dye inenergy-receiving proximity therewith.

-   Clause 40. The polymeric tandem dye according to clause 39, wherein    the polymeric dye comprises a thiophene containing co-monomer.-   Clause 41. The polymeric tandem dye according to clause 39, wherein    the polymeric dye has an absorption maximum wavelength from 440 nm    to 530 nm.-   Clause 42. The polymeric tandem dye according to clause 39, wherein    the signaling chromophore emission has a quantum yield of 0.1 or    more.-   Clause 43. The polymeric tandem dye according to clause 39, wherein    the polymeric dye has an extinction coefficient of 1×10⁶ M⁻¹ cm⁻¹ or    more.-   Clause 44. The polymeric tandem dye according to any one of clauses    39-43, wherein the multichromophore is substituted with non-ionic    side groups capable of imparting solubility in water in excess of 10    mg/mL.-   Clause 45. The polymeric tandem dye according to any one of clauses    39-44, wherein the signaling chromophore is a fluorophore.-   Clause 46. The polymeric tandem dye according to any one of clauses    39-44, wherein the signaling chromophore is a quencher.-   Clause 47. The polymeric tandem dye according to any one of clauses    39-46, wherein the signaling chromophore is selected from a    rhodamine, a coumarin, a xanthene, a cyanine, a polymethine, a    pyrene, a dipyrromethene borondifluoride, a napthalimide, a    phycobiliprotein, a peridinum chlorophyll protein, conjugates    thereof, and combinations thereof.-   Clause 48. The polymeric tandem dye according to any one of clauses    39-45 and 47, wherein the signaling chromophore is selected from    fluorescein, 6-FAM, rhodamine, Texas Red, California Red, iFluor594,    tetramethylrhodamine, a carboxyrhodamine, carboxyrhodamine 6G,    carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow,    coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy7®, Cy-Chrome, DyLight    350, DyLight 405, DyLight 488, DyLight 549, DyLight 594, DyLight    633, DyLight 649, DyLight 680, DyLight 750, DyLight 800,    phycoerythrin, PerCP (peridinin chlorophyll-a Protein), PerCP-Cy5.5,    JOE (6-carboxy-4′,5′-dichloro-2′,7′-dimelhoxyfluorescein), NED, ROX    (5-(and -6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue,    Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor®    350, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa    Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633,    Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680,    7-amino-4-methylcoumarin-3-acetic acid, BODIPY® FL, BODIPY® FL-Br2,    BODIPY® 530/550, BODIPY® 558/568, BODIPY® 564/570, BODIPY® 576/589,    BODIPY® 581/591, BODIPY® 630/650, BODIPY® 650/665, BODIPY® R6G,    BODIPY® TMR, BODIPY® TR, conjugates thereof and combinations    thereof.-   Clause 49. The polymeric tandem dye according to any one of clauses    39 to 49, wherein:

the signaling chromophore is linked to a co-monomer comprising 1% to 20%by molarity of the multichromophore; and

the polymeric dye is a conjugated polymer comprising 5 or more repeatunits.

-   Clause 50. The polymeric tandem dye according to any one of clauses    39 to 45 and 47-49, wherein the signaling chromophore emission is    1.5-fold greater or more when excited by the polymeric dye as    compared to direct excitation of the acceptor chromophore with    incident light.-   Clause 51. The polymeric tandem dye according to any one of clauses    39-50, wherein the polymeric dye comprises a terminal group -L³-Z    where L³ is a linker and Z is a specific binding member.-   Clause 52. The polymeric tandem dye according to clause 51, wherein    the linker is selected from the group consisting of an alkyl, a    substituted alkyl, an alkyl-amido, an alkyl-amido-alkyl and a PEG    moiety.-   Clause 53. The polymeric tandem dye according to clause 51, wherein    -L³-Z is described by the following structure:

wherein: q is 0 or an integer from 1-12; and Z is the specific bindingmember.

-   Clause 54. The polymeric tandem dye according to any one of clauses    51-53, wherein Z is a biomolecule.-   Clause 55. The polymeric tandem dye according to any one of clauses    51-54, wherein Z is an antibody.-   Clause 56. The polymeric tandem dye according to any one of clauses    51-54, wherein Z is an antibody fragment or binding derivative    thereof.-   Clause 57. The polymeric tandem dye according to clause 56, wherein    the antibody fragment or binding derivative thereof is selected from    the group consisting of a Fab fragment, a F(ab′)₂ fragment, a scFv,    a diabody and a triabody.-   Clause 58. A labelled specific binding member, comprising:    -   a polymeric dye having a blue excitation spectrum according to        any one of clauses 1-38; and    -   a specific binding member covalently linked to the polymeric        dye.-   Clause 59. The labelled specific binding member according to clause    58, wherein the polymeric dye comprises a thiophene containing    co-monomer.-   Clause 60. The labelled specific binding member according to any one    of clauses 58-59, wherein the polymeric dye comprises a signaling    chromophore covalently linked to the polymeric dye in    energy-receiving proximity therewith.-   Clause 61. The labelled specific binding member according to any one    of clauses 59-60, wherein the specific binding member is an    antibody.-   Clause 62. The labelled specific binding member according to any one    of clauses 59-60, wherein the specific binding member is an antibody    fragment or binding derivative thereof.-   Clause 63. The labelled specific binding member according to clause    62, wherein the antibody fragment or binding derivative thereof is    selected from the group consisting of a Fab fragment, a F(ab′)₂    fragment, a scFv, a diabody and a triabody.-   Clause 64. The labelled specific binding member according to clause    60, wherein the signaling chromophore is selected from a rhodamine,    a coumarin, a xanthene, a cyanine, a polymethine, a pyrene, a    dipyrromethene borondifluoride, a napthalimide, a phycobiliprotein,    a peridinum chlorophyll protein, conjugates thereof, and    combinations thereof.-   Clause 65. A method of evaluating a sample for the presence of a    target analyte, the method comprising:    -   (a) contacting the sample with a polymeric dye conjugate that        specifically binds the target analyte to produce a labelling        composition contacted sample, wherein the polymeric dye        conjugate comprises:        -   (i) a polymeric dye having a blue excitation spectrum            according to any one of clauses 1-38; and        -   (ii) a specific binding member covalently linked to the            polymeric dye; and    -   (b) assaying the labelling composition contacted sample for the        presence of a polymeric dye conjugate-target analyte binding        complex to evaluate whether the target analyte is present in the        sample.-   Clause 66. The method according to clause 65, wherein the polymeric    dye comprises a thiophene containing co-monomer.-   Clause 67. The method according to any one of clauses 65-66, wherein    the polymeric dye comprises a signaling chromophore covalently    linked to the multichromophore in energy-receiving proximity    therewith.-   Clause 68. The method according to any one of clauses 65-67, further    comprising contacting the sample with a second specific binding    member that is support bound and specifically binds the target    analyte.-   Clause 69. The method according to clause 68, wherein the support    comprises a magnetic particle.-   Clause 70. The method according to any one of clauses 65-69, wherein    the target analyte is associated with a cell.-   Clause 71. The method according to clause 70, wherein the target    analyte is a cell surface marker of the cell.-   Clause 72. The method according to clause 71, wherein the cell    surface marker is selected from the group consisting of a cell    receptor and a cell surface antigen.-   Clause 73. The method according to clause 70, wherein the target    analyte is an intracellular target, and the method further comprises    lysing the cell.-   Clause 74. The method according to any one of clauses 65-73, wherein    the method further comprises flow cytometrically analyzing the    fluorescently labelled target analyte.-   Clause 75. A method of labelling a target molecule, the method    comprising:

contacting the target molecule with a polymeric dye to produce alabelled target molecule, wherein the polymeric dye has a blueexcitation spectrum and comprises a conjugation tag that covalentlylinks to the target molecule.

-   Clause 76. The method according to clause 75, wherein the polymeric    dye comprises a thiophene containing co-monomer.-   Clause 77. The method according to any one of clauses 75-76, wherein    the polymeric dye comprises a signaling chromophore covalently    linked to the polymeric dye in energy-receiving proximity therewith.-   Clause 78. The method according to any one of clauses 75-77, further    comprising fluorescently detecting the labelled target molecule.-   Clause 79. The method according to any one of clauses 75-78, wherein    the conjugation tag comprises a terminal functional group selected    from an amino, a thiol, a hydroxyl, a hydrazine, a hydrazide, a    azide, an alkyne, maleimide, iodoacetyl, amine, an active ester and    a protein reactive group.-   Clause 80. The method according to any one of clauses 75-79, wherein    the target molecule is a specific binding member.-   Clause 81. The method according to clause 80, wherein the specific    binding member is an antibody.-   Clause 82. The method according to clause 80, wherein the specific    binding member is an antibody fragment or binding derivative    thereof.-   Clause 83. The method according to clause 82, wherein the antibody    fragment or binding derivative thereof is selected from the group    consisting of a Fab fragment, a F(ab′)₂ fragment, a scFv, a diabody    and a triabody.-   Clause 84. A flow cytometric system, comprising:    -   a flow cytometer comprising a flow path;    -   a composition in the flow path, wherein the composition        comprises:        -   a sample; and        -   a labelled specific binding member comprising:            -   a polymeric dye having a blue excitation spectrum                according to any one of clauses 1-38; and            -   a specific binding member that specifically binds a                target analyte and is covalently linked to the polymeric                dye.-   Clause 85. The system according to clause 84, wherein the polymeric    dye comprises a thiophene containing co-monomer.-   Clause 86. The system according to any one of clauses 84-85, wherein    the labelled specific binding member further comprises a signaling    chromophore covalently linked to the polymeric dye in    energy-receiving proximity therewith.-   Clause 87. The system according to any one of clauses 84-86, wherein    the composition further comprises a second specific binding member    that is support bound and specifically binds the target analyte.-   Clause 88. The system according to clause 85, wherein the support    comprises a magnetic particle.-   Clause 89. The system according to any one of clauses 84-88, wherein    the sample comprises a cell.-   Clause 90. The system according to clause 89, wherein the target    analyte is a cell surface marker of the cell.-   Clause 91. The system according to clause 90, wherein the cell    surface marker is selected from the group consisting of a cell    receptor and a cell surface antigen.-   Clause 92. A kit comprising: a polymeric dye having a blue    excitation spectrum; and a container.-   Clause 93. The kit according to clause 92, wherein the polymeric dye    comprises a thiophene containing co-monomer.-   Clause 94. The kit according to any one of clauses 92-93, further    comprising one or more components selected from the group consisting    of a polymeric tandem dye, a fluorophore, a specific binding member,    a specific binding member conjugate, a cell, a support, a    biocompatible aqueous elution buffer and instructions for use.-   Clause 95. The kit according to any one of clauses 92-94, wherein    the polymeric dye is described by any one of clauses 2-53.-   Clause 96. The kit according to any one of clauses 92-95, wherein    the polymeric dye is covalently linked to a specific binding member.-   Clause 97. The kit according to clause 96, wherein the specific    binding member is an antibody.-   Clause 98. The kit according to clause 96, wherein the specific    binding member is an antibody fragment or binding derivative    thereof.-   Clause 99. The kit according to clause 98, wherein the antibody    fragment or binding derivative thereof is selected from the group    consisting of a Fab fragment, a F(ab′)₂ fragment, a scFv, a diabody    and a triabody.-   Clause 100. The kit according to any one of clauses 92-99, wherein    polymeric dye further comprises an acceptor signaling chromophore    covalently linked to the polymeric due in energy-receiving proximity    therewith.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the following.

1. A water solvated polymeric dye having a blue excitation spectrum.
 2. The polymeric dye according to claim 1, wherein the polymeric dye comprises a thiophene containing co-monomer.
 3. The polymeric dye according to claim 1, wherein the excitation spectrum of the polymeric dye has a full width at half maximum (FWHM) that is 100 nm or less in width.
 4. The polymeric dye according to claim 1, wherein the polymeric dye comprises a conjugated segment having the structure of formula (I):

wherein: each M¹, M² and M³ is independently an aryl or heteroaryl co-monomer wherein at least one of M¹, M² and M³ is a thiophene containing co-monomer; Z¹ is a chemoselective functional group or a linked acceptor chromophore; each Z⁵ and Z⁶ are independently CR or N wherein R is selected from H, halogen, alkoxy, substituted alkoxy, alkyl and substituted alkyl; G¹ and G² are each independently selected from a terminal group, a π conjugated segment, a linker and a linked specific binding member; Y¹, Y² and Y³ are independently C(R³)₂, —C(R³)₂C(R³)₂—, NR³, Si(R³)₂ or Se; each R³ is independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido and a WSG; a-i are independently 0 or 1; each n, m and o are independently 0 or an integer from 1 to 10,000; and p is an integer from 1 to 100,000.
 5. The polymeric dye according to claim 4, wherein: a+c≦1; d+f≦1; g+i≦1; b+e+h≧1; and n+m+o≧1.
 6. The polymeric dye according to claim 4, wherein the polymeric dye has the structure of formula (Ic):


7. The polymeric dye according to claim 4, wherein the polymeric dye has the structure of formula (IIIb):

wherein n is an integer from 1 to 100,000.
 8. The polymeric dye according to claim 4, wherein the polymeric dye has the structure of formula (IVb):

wherein: M¹ and M² are each independently a thiophene containing co-monomer; each n and m are independently an integer from 1 to 10,000; p is an integer from 1 to 100,000.
 9. The polymeric dye according to claim 6, wherein the polymeric dye has: a) the structure of formula (Vb):

wherein: M¹ and M³ are each independently a thiophene containing co-monomer; each n and each o are independently an integer from 1 to 10,000; and p is an integer from 1 to 100,000; or b) the structure of formula (VIb):

wherein: M¹ and M² are each independently a thiophene containing co-monomer; each n and each m are independently an integer from 1 to 10,000; Z¹ is a linked chemoselective functional group or a linked signaling chromophore; and p is an integer from 1 to 100,000.
 10. The polymeric dye according to claim 9, wherein Y³-Z¹ is C(R³)(T¹-Z¹), wherein Z¹ is a linked signaling chromophore and T¹ is a linker.
 11. The polymeric dye according to claim 9, wherein: Y¹ and Y² are each independently C(R³)₂; and M¹, M² and M² are the same thiophene containing co-monomer.
 12. The polymeric dye according to claim 2, wherein the thiophene-containing co-monomer is of formula (VII), (VIII) or (IX):

wherein: A1 and A2 are each a fused monocyclic or bicyclic group each R³ is independently selected from H, amino, substituted amino, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido, substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy, WSG and -T²-Z², wherein Z² is a chemoselective functional group or a linked signaling chromophore, and T² is a linker; n is 0, 1, 2, 3 or 4; and each * is a site for covalent attachment to the unsaturated backbone of a conjugated polymer.
 13. The polymeric dye according to claim 12, wherein the thiophene-containing co-monomer is described by one of formula (X)-(XVII):

wherein: X¹ is CR³, NR³, O or S; X² is N, O or S, wherein when X² is N, each

is a double bond and when X² is O or S, each

is a single bond; R¹ and R² together form a 5- or 6-membered fused aryl or heteroaryl ring which is optionally substituted with one or more R³ groups; and Y⁴ is NR³, C(R³)₂ or Si(R³)₂.
 14. The polymeric dye according to claim 13, wherein the thiophene-containing co-monomer is described by one of the following structures (a) to (q):

wherein: each R⁴ is independently selected from H, amino, substituted amino, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido, substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy and -T²-Z², wherein Z² is a chemoselective functional group or a linked acceptor chromophore, and T² is a linker; and each s is 0 or an integer from 1 to
 50. 15. The polymeric dye according to claim 12, wherein the thiophene-containing co-monomer is described by one of the following structures (ba) to (bu):


16. The polymeric dye according to claim 4, wherein co-monomers M¹-M³ are independently selected from a co-monomer of formulae (XVIII) and (XIX):

wherein: R¹ and R² together form a 5- or 6-membered fused aryl or heteroaryl ring which is optionally substituted with one or more R³ groups; and each R³ is independently selected from H, amino, substituted amino, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido, substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy and -T²-Z², wherein Z² is a chemoselective functional group or a linked acceptor chromophore, and T² is a linker.
 17. The polymeric dye according to claim 4, wherein co-monomers M¹-M³ are independently selected from a co-monomer of formulae (e)-(i):

wherein: each R⁴ is independently selected from H, amino, substituted amino, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido, substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy and -T²-Z², wherein Z² is a chemoselective functional group or a linked acceptor chromophore, and T² is a linker; and each s is 0 or an integer from 1 to
 50. 18. The polymeric dye according to claim 12, wherein each R³ and each R⁴ is independently selected from H, substituted aryl, alkyl and the following structures:

wherein: each T⁵ is independently an optional linker; and each s is 0 or an integer from 1 to
 50. 19. A polymeric tandem dye comprising: a polymeric dye having a blue excitation spectrum according to claim 1; and a signaling chromophore covalently linked to the polymeric dye in energy-receiving proximity therewith.
 20. A labelled specific binding member, comprising: a polymeric dye having a blue excitation spectrum according to claim 1; and a specific binding member covalently linked to the polymeric dye. 